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DIAGNOSTIC METHODS 



WEBSTER 



DIAGNOSTIC METHODS 

CHEMICAL, BACTERIOLOGICAL 
AND MICROSCOPICAL 



A Text-book for Students and Practitioners 



RALPH Wr WEBSTER, M. D., Ph. D. 

ASSISTANT PROFESSOR OF PHARMACOLOGICAL THERAPEUTICS AND INSTRUCTOR IN MEDICINE IN 

RUSH MEDICAL COLLEGE, UNIVERSITY OF CHICAGO; DIRECTOR OF CHICAGO 

LABORATORY, CLINICAL AND ANALYTICAL 



SIXTH EDITION, REVISED AND ENLARGED 

WITH 37 COLORED PLATES 

AND 170 OTHER ILLUSTRATIONS 



PHILADELPHIA 

P. BLAKISTON'S SON & CO. 

1012 WALNUT STREET 



FiBST Edition, Copyright, 1909, by P. Blakiston's Son & Co. 
Second Edition, Copyright, 191 2, by P. Blakiston's Son & Co. 
Third Edition, Copyright, 1913, by P. Blakiston's Son & Co. 
Fourth Edition, Copyright, 1914, by P. Blakiston's Son & Co. 
Fifth Edition, Copyright, 1916, by P. Blakiston's Son & Co. 
Sixth Edition, Copyright, 1920, by P. Blakiston's Son & Co. 



A«fi (0 f920 



IE MAPLE PRESS YORK PA 



©CI.A570993 






TO THE MEMORY 

' OF 

MY FATHER 
DR. JOHN RANDOLPH WEBSTER 

THIS VOLUME IS 

LOVINGLY DEDICATED 



PREFACE TO THE SIXTH EDITION. 



During the few years since the appearance of the last edition, original 
work along all lines has suffered from the absence of many of our associates 
in the service. However, quite an amount of material, of sufficient impor- 
tance to warrant inclusion in this edition, has been forthcoming from various 
sources. 

Among these additions will be found quite a number of new methods from 
the laboratory of Folin such as Folin and Bell's direct nesslerization method 
for ammonia in urine; Folin and Denis' direct nesslerization method for total 
N in urine, and their method for lactose in milk; Folin and McEllroy's test 
for sugar in the urine; Folin and Peck's method for sugar in the urine; 
Folin and Wright's simphfied Kjeldahl method; FoHn and Wu's system of 
blood analysis, which includes methods for creatin and creatinin in blood, 
non-protein nitrogen in blood, sugar in blood, urea in blood, uric acid in 
blood, and uric acid in urine; Folin and Youngburg's direct nesslerization 
method for urea in urine. The subject of functional renal diagnosis has been 
extensively enlarged and a full discussion of Mosenthal's Test Meal for Renal 
Function has been included. The section dealing with the reaction of the 
blood has been entirely rewritten to bring it up to the present day concep- 
tion of Hydrogen-ion concentration; in this section the following methods 
have been included, as measures of the reserve alkalinity and the total Ph 
of the blood: Van Slyke and CuUen's method for CO2 combining power of 
the plasma; Van Slyke, Stillman, and Cullen's titration method for plasma 
bicarbonate; electrometric methods for determination of Ph; Levy, Rown- 
tree and Marriott's method for hydrogen ion concentration; Marriott's 
method for alkali reserve. In the discussion of the parasitology of the blood, 
the subject of Infectious Jaundice has been introduced, a full account of the 
Leptospira icterohsemorrhagica being given. It has been thought advisable 
to include among the serum reactions for syphilis, the Coagulo Reaction of 
Hirschfeld and Klinger, as this has great promise. In the section on Clinical 
Bacteriology, the discussion has now included the Gas Bacillus of Welch, 
which has assumed considerable importance in the study^of wound infections 
during the war. Throughout the text the subject-matter has been brought 
thoroughly up to date by reference to the literature of the subjects treated. 

The author wishes to express his thanks for the more than cordial way 

in which the previous editions of this book have been received and to thank 

his reviewers and friends for words of criticism and praise. It is hoped 

that this new edition may prove even more acceptable than have former 

ones. 

Ralph W. Webster. 
25 E. Washington St., Chicago. 



PREFACE TO THE FIFTH EDITION 



During the last year, investigation along all scientific lines has been greatly 
curtailed for self-evident reasons. However, sufficient new material, both as 
regards methods and general points of attack, is at hand to warrant quite 
extensive changes in the subject matter of this work. 

Among the additions will be found Petroff's method of obtaining pure cul- 
tures of tubercle bacilli; a discussion of the Endameba gingivalis; description 
and methods of study of Bacillus rhinitis; Wolff- Junghans' test for gastric 
carcinoma; Wagner's ''dry test" for occult blood; Phenoltetrachlorphthalein 
test of hepatic activity; Urease method for urea; Folin's new method for crea- 
tinin in urine; Benedict and Murlin's method for amino-acids in urine; Folin 
and Denis' micro-chemical methods for non-protein nitrogen, urea and uric 
acid in the blood; Schick diphtheria toxin reaction; Bronfenbrenner's modifi- 
cation of Abderhalden's test; and tests of blood before transfusion. The 
subject matter throughout the text has been brought up to date by the inclu- 
sion of a large number of references to the original literature. 

The author wishes to express his extreme gratification at the reception of 
his former editions and, also, his deepest thanks for any and all criticisms 
which he has obtained from his reviewers and friends. He desires, especially, 
to acknowledge his indebtedness to Dr. F. B. Moorehead for his kindly criti- 
cism of the section on Endameba gingivalis. 

Ralph W. Webster. 

25 E. Washington St., Chicago. 



PREFACE TO THE FOURTH EDITION. 



Although less than a year has elapsed since the appearance of the third edi- 
tion of this work, the author has felt justified in adding considerable new 
material in this revision. This has come about largely through the sugges- 
tions of teachers and general practitioners regarding the inclusion of certa^in 
subjects not hitherto discussed. 

Thus, the new matter embraces, among other subjects, a discussion of the 
Negri bodies recently successfully cultivated by Noguchi; Weisz's test for 
urochromogen; Dorrance's method for coagulation of the blood; Kowarsky's 
method for' glucose in the blood; Levaditi and Manouelian's as well as Nogu- 
chi's methods for staining spirochaete in tissue; Lange's colloidal-gold test for 
congenital syphilis; a discussion of the organism of anterior poliomyelitis 
recently cultivated by Flexner and Noguchi. The Wassermann test has 
been brought up to date by inclusion of considerable new matter; while 
the complement-fixation test in gonorrhea has been elaborated. The Her- 
man-Perutz reaction for syphilis is fully described. The application of 
Abderhalden's dialyzation method to the diagnosis of cancer and dementia 
praecox has been freely discussed. Further, an entirely new chapter on 
Clinical Bacteriology has been added, in which are discussed the differen- 
tiation of the more important pathogenic organisms and, also, the methods 
of preparation of vaccines. The subject matter throughout the text has 
been brought up to date by the inclusion of a large number of references to 
the original literature. 

The author wishes to express his great satisfaction at the reception of the 
third edition of his work and, also, his full appreciation of the kind sugges- 
tions offered him regarding the general matter ,of the book. He wishes, 
especially, to thank his colleagues Drs. T. L. Dagg and C. C. Croy for their 
many valuable suggestions and criticism. 

Ralph W. Webster. 

25 E. Washington St., Chicago. 



PREFACE TO THE FIRST EDITION. 



In the present work the writer has attempted to bring together, for the 
use of the student and practitioner, the generally accepted facts regarding the 
various phases of clinical medicine, which may be rather more closely studied 
by the application of laboratory methods than without their use. It is to be 
especially emphasized that laboratory work must go hand in hand with the 
more direct clinical examination of the patient, as the former can be inter- 
preted only in the light of the latter. While it is true that, in some cases, 
the laboratory findings may be of even more value than those of physical ex- 
amination, yet it is to be understood that the function of the Clinical Labora- 
tory is, more largely, perhaps, an accessory one, the application of methods of 
physical diagnosis usually pointing out the way toward a successful solution 
of the clinical problem by special laboratory methods, which yield numerous 
confirmatory or differential points not at all clearly defined by the methods of 
direct clinical examination. 

The aim of the author has been to present the direct bearing of the various 
methods outlined upon the clinical history of the case and to point out the 
special interpretation of the findings in any given examination. Particular 
attention has been directed to the selection of methods, both for the simpler 
clinical and more complex scientific requirements. As no method, no matter 
how exact it may be or how sound its basic principle, will yield reliable re- 
sults in the hands of the inexperienced, the writer has paid much attention to 
the details of such methods and has endeavored to direct the thought of the 
worker to the possible obstacles to be overcome before he is able properly to 
perform these examinations and interpret his results. 

The writer makes no claim for originality except, possibly, in the matter of 
arrangement of subject-matter and selection of methods and ideas, which 
have been established by others after years of earnest research. His endeavor 
has been to be as catholic as possible in his reading and as selective as his 
judgment permitted, to the end that the student or practitioner be saved the 
burden of sifting the wheat from the chaff. 

Numerous text-books, monographs, and special articles have been freely 
used in the preparation of the text, the writer attempting in each case to give 
due credit for such reference. It is possible that some direct use of material 
has been made which has not received deserved recognition. If so, the writer 
here acknowledges his indebtedness to them as well as to those whom he has 
directly quoted. 

It has seemed desirable to omit extensive reference to the literature, as a 
bibliography to be of working value must be much more extensive and com- 



XIV PREFACE 

plete than is possible within the scope of this book. The writer has, however, 
inserted at the end of each chapter a list of the more important larger works 
which he has found useful in correlating the general subjects included in the 
several sections. 

In conclusion, the writer wishes to express his deep obligation to his col- 
leagues for their many valuable suggestions as to the subject-matter of the 
text: to Prof. W. S. Haines for assistance in revising the section on Urine; to 
Dr. J. M. Washburn for many additions and revisions of the section on Blood; 
to Dr. O. J. West for many practical points throughout the whole work; to 
Dr. W. A. Pusey for photographs of Blastomycetes under various conditions; 
to Dr. Brown Pusey for slides showing various organisms in the conjunctival 
exudates; and to Dr. N. Gildersleeve, of the University of Pennsylvania, for 
illustrations of megalosporon and microsporon. Further, the writer wishes 
to express his thanks to Miss Hill for the excellent work done by her in pre- 
paring the original drawings which appear throughout the work. 

Ralph W. Webster. 



TABLE OF CONTENTS 



CHAPTER I 
The Sputum 

Page 

I. General Considerations i 

II. Physical and Chemical Characteristics • • • 2 

Amount 2 

Consistency 2 

Reaction 3 

Color 3 

Odor 4 

Character 4 

Chemistry 5 

III. Macroscopic Examination • 6 

Cheesy masses 6 

Dittrich's plugs 6 

Curschmann's spirals 7 

Fibrinous casts 8 

Concretions 8 

Bronchioliths . ^ 8 

Pneumoliths 9 

Echinococcus membranes 9 

Foreign bodies 9 

IV. Microscopic Examination 9 

Pus-cells 10 

Red blood-cells Ii 

Epithelial cells 11 

Elastic tissue 12 

Crystals 13 

Bacteria 14 

Saprophytes 14 

Pathogenic types 17 

Tubercle bacillus . 17 

Lepra bacillus 24 

Smegma bacillus ' . . 24 

Timothy bacillus 25 

Pneumococcus 25 

Friedlander's bacillus 26 

Influenza bacillus 26 

Bacillus pertussis 26 

Bacillus typhosus 27 

Staphylococcus and streptococcus pyogenes 27 

Bacillus pestis 27 

Bacillus anthracis 27 

Bacillus mallei 28 

Actinomyces hominis 28 

XV 



XVI TABLE OF CONTENTS 

Page 

Animal parasites , 29 

Amebae 29 

Flagellates 29 

Cestodes 30 

Trematodes 30 

V. The Sputa in Disease 30 

Pulmonary tuberculosis • 30 

Croupous pneumonia 31 

Broncho-pneumonia 32 

Acute bronchitis 32 

Chronic bronchitis 32 

Simple type 32 

Putrid type 33 

Fibrinous type 33 

Bronchial asthma 33 

Influenza 33 

Gangrene of the lung 34 

Abscess of the lung ' 34 

Perforating empyema 34 

Pneumonoconioses 34 

CHAPTER II 
Oral, Nasal, Aural and Conjunctival Secretions 

I. Oral Secretion 35 

General considerations 35 

Microscopic examination 36 

Pathologic changes 37 

Pharyngomycosis leptothrica 38 

Diphtheria 38 

Vincent's angina 40 

Streptococcic sore throat 41 

Gonorrheal stomatitis 41 

Thrush 41 

Oral endamebiasis 42 

II. Nasal Secretion 44 

General considerations 44 

Pathologic changes 45 

Rhinitis " 45 

Hay fever 46 

Meningitis 46 

III. Aural Secretion 47 

General considerations 47 

Pathologic changes 47 

IV. Conjunctival Secretions 47 

General considerations 47 

Pathologic changes 48 

Diphtheritic conjunctivitis 48 

Infectious conjunctivitis 48 

Gonorrheal conjunctivitis 48 

Trachoma 49 

Vernal conjunctivitis 49 



TABLE OF CONTENTS XVll 

CHAPTER III 
Gastric Contents 

Page 

I. General Considerations 50 

11. Methods of Obtaining Gastric Contents 52 

Stomach-tube 52 

Test meals 54 

• Ewald meal 54 

Boas meal 55 

Riegel meal 55 

Fischer meal 55 

Salzer meal 56 

Sahli meal 56 

III. AIacroscopic Examination 57 

Amount 57 

Color * 58 

Odor 59 

Consistency 59 

Contents from fasting stomach 60 

Vomitus 60 

Contents after test meals . 61 

IV. Microscopic Examination 62 

General 62 

Food remnants 62 

Boas-Oppler bacillus 62 

Sarcinae ventriculi 63 

Protozoa 63 

Tissue fragments 63 

Crystals 63 

V. Chemical Examination. . 63 

General 64 

Total acidity 64 

Free hydrochloric acid 65 

Qualitative tests 66 

Topfer's test 66 

Gunzburg's test 66 

Boas' test 67 

Tropeolin test 67 

Quantitative tests 67 

Mintz's method 68 

Topfer's method 68 

Amount of free hydrochloric acid 69 

Euchlorhydria 70 

, Hypochlorhydria 70 

Ana-chlorhydria 70 

Hyperchlorhydria 71 

Combined hydrochloric acid , 71 

Method of Martins and Luttke 71 

Method of Topfer 72 

Hydrochloric acid deficit 73 

Organic acids 73 

Total organic acids 73 

Lactic acid 74 

Ujffelmann's test 75 



XVlll TABLE OF CONTENTS 

Page 

Kelling's test. 75 

Strauss' method 75 

Butyric acid 76 

Acetic acid 77 

Gastric ferments 77 

Pepsin 77 

Qualitative methods 78 

Quantitative examination 79 

Hammerschlag's method 79 

Mett's method 79 

Method of Thomas and Weber 80 

Chymosin 81 

Leo's method 81 

Riegel's method ; ,. . . 81 

Lipase 82 

Products of protein digestion 82 

Products of carbohydrate digestion 82 

Blood 83 

Gases 83 

Function of the stomach and contents 84 

VI. Motility of the Stomach 84 

Leube's method 85 

Boas' method 85 

Method of Ewald and Sievers 85 

Winternitz' test 86 

Vn. Absorptive Power of the Stomach 86 

Potassium iodide test 86 

VIII. Indirect Examination of the Stomach Contents 87 

Gunzburg's method 87 

Sahli's desmoid reaction 87 

IX. Gastric Juice in Disease 88 

Hyperchlorhydria 88 

Hypersecretion 89 

Achylia gastrica 89 

Acute gastritis 90 

Chronic gastritis 90 

Nervous dyspepsia 90 

Ulcer of the stomach 91 

Carcinoma of the stomach 91 

Salomon's test 93 

Neubauer and Fischer's test 93 

Wolff and Junghans' test 95 

CHAPTER IV 

The Feces 

I. General Considerations . 99 

Normal feces 99 

Diet of Schmidt and Strasburger 100 

Diet of FoHn loi 

Obtaining intestinal juice loi 

Functions of the intestinal juice 102 

Estimation of intestinal digestion 103 



TABLE OF CONTENTS XIX 

Page 

II. Macroscopic Examination 103 

Method 103 

Amount 104 

Consistency and form 105 

Odor 106 

Color 106 

Blood 108 

Mucus Ill 

Pus 113 

Food remnants .■ 113 

Protein residues 114 

Pat residues 115 

Carbohydrate residues 116 

Biliary constituents 117 

Intestinal sand and concretions . • ... 118 

Tissue fragments 118 

III. Microscopic Examination 119 

Technic 119 

Morphological elements 119 

Crystals .120 

IV. Chemical Examination. 121 

Reaction 121 

Total solids 121 

Total nitrogen 122 

Pat 123 

Carbohydrates 123 

Phenoltetrachlorphthalein test ^ 125 

V. Bacteriology of the Feces . . . 128 

Technic 128 

Cholera spirillum 129 

Typhoid bacillus 130 

Method of Drigalski and Conradi 131 

Method of Kendall and Day 132 

Bacillus of Dysentery 132 

Tubercle bacillus . . 133 

VI. Parasitology of the Feces 133 

Technic 133 

Protozoa 134 

Rhizopoda 135 

Amebina 135 

Ameba coli 135 

Entameba coli 139 

Sporozoa . 139 

Cocci dium hominis 139 

Flagellata 140 

Trichomonas intestinalis . 140 

Cercomonas hominis 140 

Megastoma entericum 140 

Infusoria 141 

Balantidium coli 141 

Entozoa 141 

Platodes 141 

Cestodes 141 

Taeniidse 143 



XX TABLE OF CONTENTS 

Page 

Taenia solium 143 

Tasnia saginata 143 

Tasnia cucumerina 144 

Tasnia nana 144 

Tasnia diminuta 145 

Taenia echinococcus 145 

Bothriocephaloidea 146 

Bothriocephalus latus 146 

Dibothriocephalus cordatus 147 

Bothriocephalus sp. Ijima et Kurimoto .... 147 

Trematodes 147 

Nematodes 148 

Ascaridas 148 

Ascaris lumbricoides 148 

Ascaris mystax 149 

Oxyuris vermicularis 149 

Angiostomidse 150 

Strongyloides intestinalis 150 

Trichotrachelidas ' .... 152 

Trichiuris trichiura 152 

Trichinella spiralis 152 

Strongylidae 154 

Uncinaria duodenalis 154 

Uncinaria Americana 155 

Pseudo-parasites 156 

CHAPTER V 

Parasites 

I. General Considerations 159 

II. Trematodes 159 

Fasciolidae 160 

Fasciola hepatica 160 

Fasciolopsis Buski 161 

Opisthorchis felineus 162 

Opisthorchis sinensis 162 

III. Nematodes 163 

Eustrongylus gigas 163 

IV. Parasites of the Skin 164 

Arthropoda 164 

Arachnoidea 165 

Sarcoptes scabiei ■ . . 165 

Demodex folliculorum 165 

Leptus autumnalis . . 165 

Insecta * 166 

Hemiptera 166 

Pedi cuius capitis 166 

Pediculus vestimenti 166 

Pedi cuius pubis 166 

Cimex lectularius 167 

Diptera 168 

Pulex irritans 168 

Pulex penetrans 168 



TABLE OF CONTENTS XXI 

Page 

Vegetable Parasites i68 

Achorion Schonleinii i68 

Trichophyton megalosporon endothrix 169 

Microsporon Audouini 170 

Microsporon furfur 170 

Microsporon minutissimum 172 

Blastomycetes 172 

Sporothrix Schenckii 174 

Negri Bodies 175 

CHAPTER VI 
The Urine 

I. General Considerations 178 

Collection and preservation of the urine 179 

II. Physical Properties 180 

Quantity 180 

Polyuria 181 

Oliguria 182 

Anuria 183 

Appearance 183 

Color 184 

Odor 187 

Reaction 187 

Folin's method for total acidity 188 

Free mineral and organic acidity 190 

Specific gravity 192 

Technic 192 

Rough estimate of total solids . . , 193 

Optical activity 195 

III. Chemical Properties 195 

Normal composition 195 

Total solids and total ash 196 

Inorganic constituents 197 

Chlorids 197 

Estimation of the chlorids 200 

Quantitative determination 200 

Volhard's method 200 

Purdy's centrifugal method 203 

Phosphates 203 

Estimation of phosphates 206 

Quantitative determination 207 

Uranium method 207 

Total phosphoric acid 210 

Purdy's centrifugal method 210 

Sulphur compounds 210 

Preformed sulphates ....211 

Ethereal sulphates 212 

Neutral sulphur 213 

Estimation of total sulphur ...' 213 

Folin's method 213 

Determination of total sulphates 214 

Folin's method 214 



XXU TABLE OF CONTENTS 

Page 

Determination of inorganic sulphates 215 

Purdy's centrifugal method 215 

Carbonates 216 

Sodium and potassium ....216 

Calcium and magnesium 216 

Iron 218 

Organic constituents 218 

Nitrogenous bodies 218 

Total nitrogen 218 

Kjeldahl's method 222 

Folin and Wright's Simplified Kjeldahl method . 224 
'^ Folin and Denis' Direct Nesslerization method . 225 

Urea 228 

Determination of urea 229 

Knop-Hiifner method 230 

Doremus ureometer 230 

Folin's method 232 

Morner-Sjoqvist method. .• 233 

Urease method 234 

Folin and Youngburg's Direct Nesslerization 

method 236 

Ammonia 237 

Quantitative determination 238 

Method of Schlosing 239 

Folin's method . 240" 

Folin and Bell's Direct Nesslerization method 241 

Formalin method 242 

Uric acid 242 

Quantitative determination 246 

Folin and Shaffer's method 246 

Salkowski-Ludwig method 247 

Method of Rudisch and Kleeberg 248 

Folin and Wu's Colorimetric method ... 250 

Ruhemann's method 251 

Purin bases 252 

Creatinin 253 

Qualitative tests 255 

Weyl's test 255 

Jafle's test 256 

Quantitative determination 256 

Folin and Shaffer's methods 256 

Undetermined nitrogen 259 

Amino acids 259 

Hippuric acid 261 

Oxyproteic and alloxyproteic acids 261 

Allantoin 261 

Fatty acids 262 

Oxalic acid ^ '. . . 263 

Quantitative determination 264 

Baldwin's method 264 

Ferments 264 

Pepsin 265 

Diastase 265 

Lipase 265 



TABLE OF CONTENTS XXlll 

Page 

Mucin-like bodies 265 

Mucin • 265 

Nucleo-albumin 266 

Pigments and chromogens 267 

Urochrome 267 

Uroerythrin 268 

Urobilin 268 

Indican . 270 

Tests for indican 271 

Jaffe's test 271 

Obermayer's test 271 

Rosenbach's test 272 

Quantitative determination 272 

Wang's method 272 

Polin's method . 273 

Uroroseinogen 274 

Abnormal Composition ......: 274 

Proteins '. 274 

Serum-albumin. 274 

Albuminuria 275 

Functional 275 

Febrile .278 

Traumatic 279 

Hematogenous 279 

Toxic . . 279 

Neurotic 279 

With definite renal lesions 279 

Qualitative tests 280 

Heat and acid test 281 

Heller's nitric acid test 282 

Ferrocyanide test 285 

Sulpho-salicylic acid test 285 

Spiegler's test 285 

Quantitative methods 286 

Scherer's method 286 

Esbach's method 287 

Method of Tsuchiya 287 

Purdy's centrifugal method 288 

Removal of albumin 288 

Serum-globuHn 289 

Qualitative tests 289 

Quantitative method 290 

Proteoses 290 

Primary proteoses 290 

Bence- Jones' protein 290 

Secondary proteoses 293 

Tests 293 

Bang's method 293 

Clinical significance 294 

Peptone . 295 

Hemoglobin 295 

Heller's test 296 

Donogany's test 297 

Fibrin . 297 



XXIV TABLE OF CONTENTS 

Page 

Carbohydrates 298 

Glucose 298 

Glycosuria 298 

Qualitative tests 303 

Trommer's test 303 

Benedict's test 305 

Folin and McEllroy's test 305 

Fehling's test . . . »^ . 306 

Haines' test 306 

Almen-Nylander's test : . . 308 

Fermentation test. .^ 309 

Phenyl-hydrazin test 310 

Quantitative tests 312 

Folin and Peck's method 312 

Bang's method 313 

Purdy's method 315 

Haines' method 316 

Polariscopic method 317 

Fermentation method 320 

Roberts' method 321 

Levulose 322 

Levulosuria 322 

SeliwanofE's test 323 

Phenyl-methyl-hydrazin test 323 

Pentose 325 

Pentosuria 325 

Qualitative tests 326 

Tollen's reaction 326 

Orcin test 327 

Quantitative test 327 

Diphenyl-hydrazin method 327 

Cammidge*s reaction 328 

Lactose 329 

Lactosuria 329 

Rubner's test . 330 

Maltose 330 

Maltosuria 330 

Glycuronic acid 331 

Neuberg's quantitative method 334 

Acetone bodies 334 

Acetone 337 

Qualitative tests 338 

Legal's test 338 

Lieben's test 338 

Gunning's test 338 

Frommer's test 339 

Quantitative methods 339 

Huppert-Messinger method 339 

Folin's method 341 

Diacetic acid 341 

Qualitative tests 342 

Gerhardt's test 342 

Arnold's test 342 



* TABLE OF CONTENTS XXV 

Page 

Lipliawsky's test 343 

/3-oxybutyric acid 343 

Quantitative determination 344 

Black's method ■ 344 

Shaffer's method 345 

Abnormal pigments 346 

Blood pigments 346 

Hemoglobin 346 

Hematoporphyrin 346 

Biliary pigments 346 

Qualitative tests 347 

Smith's test 347 

Gmelin's test 348 

Rosenbach's test 348 

Nakayama's test 348 

Hammarsten's test 348 

Bile acids 349 

Hay's test 349 

Oliver's test 349 

Melanin 350 

Phenol derivatives 350 

Alkapton 350 

Ehrlich's diazo reaction 352 

Russo's reaction 353 

Dimethyl-amino-benzaldehyd reaction 354 

IV. Microscopic Examination 354 

. Unorganized sediments 356 

Those appearing in acid urine 356 

Uric acid 356 

Sodium acid urate 357 

Potassium acid urate 358 

Xanthin 359 

Calcium oxalate 359 

Cystin " 360 

Cystinuria 360 

Leucin. 361 

Tyrosin 362 

Calcium sulphate 363 

Bilirubin 363 

Hippuric acid 363 

Neutral calcium phosphate '' 364 

Fat 364 

Chyluria 364 

Those appearing in alkaline urine 365 

Ammonium urate 365 

Calcium tri-phosphate 365 

Magnesium phosphate 366 

Magnesium-ammonium phosphate 366 

Calcium carbonate . 367 

Organized sediments 367 

Mucoid material 367 

Epithehal cells ^ . 367 

Pus-cells 369 

Pyuria 369 



XXVI TABLE OF CONTENTS 

Page 

Vitali's test 371 

Donne's test 372 

Enumeration of pus-cells 372 

Red blood-cells 372 

Hematuria 372 

Casts 374 

True casts 374 

Hyaline casts 374 

Granular casts 375 

Waxy casts 376 

Fibrinous casts 377 

Epithelial casts 377 

Fatty casts 377 

Blood-casts 377 

Pus-casts 377 

Cylindroids 378 

Pseudo-casts 379 

Cylindruria 379 

Spermatozoa . 381 

Tissue fragments 381 

Bacteria 381 

Bacilluria 384 

Parasites .* 384 

V. Calculi 385 

Uric acid calculi 386 

Calcium oxalate calculi 386 

Heller's table for analysis 387 

Phosphatic calculi 388 

Calcium carbonate calculi 388 

Cystin calculi 388 

Xanthin calculi) 388 

Urostealith calculi 388 

VI. Functional Diagnosis 388 

Cryoscopy 390 

Electric conductivity 390 

Mosenthal's Test Meal for Renal Function 391 

Phloridzin test 394 

Phenolsulphonephthalein test 394 



CHAPTER VII 

Secretion of the Genital Organs 

I. Male Secretions •. 398 

General considerations 398 

Microscopic examination 398 

Pathologic variations 400 

Medico-legal aspects 401 

Florence's test 401 

Barberio's test 402 

II. Female Secretions 402 

Vaginal secretions 402 

Microscopic examination 4^3 

Pathology 4^3 



TABLE OF CONTENTS XXVll 

Page 

Blenorrhea 403 

Purulent secretions 404 

Fetid secretions 404 

Uterine secretions 405 

Menstruation 405 

The lochia 405 

Amniotic fluid 405 

Abortion 406 

Vesicular mole 406 

Carcinoma 407 

CHAPTER VIII 

The Blood 

I. General Considerations 408 

II. Physiology and Chemistry .' . 409 

Blood formation and blood-forming organs 409 

Total volume of blood 410 

Volume relations of cells to plasma 413 

Methods of obtaining blood 415 

Physical properties : 416 

Color 417 

Odor 418 

Reaction. 418 

1. Van Slyke and Cullen's Method for CO2 422 

2. Van Slyke, Stillman and Cullen's Titration Method. . 426 

3. Electrometric Methods for Ph 428 

4. Colorimetric Methods for Ph 429 

(a) Levy, Rowntree and Marriott's Method .... 433 

(b) Marriott's Method for Alkah Reserve 436 

Specific gravity . 437 

Viscosity 439 

Coagulation 440 

Osmotic pressure and cryoscopy 443 

Electric conductivity 445 

Chemical properties 446 

Total solids 448 

Blood pigments 448 

Hemoglobin 448 

Pseudo-hemoglobin 450 

Oxy-hemoglobin 450 

Met-hemoglobin 450 

Carbon monoxid-hemoglobin 451 

Carbon-dioxid-hemoglobin 451 

Siilph-hemoglobin 451 

Decomposition products 452 

Hematin 452 

Hematoporphyrin 453 

Hematoidin 453 

Hemosiderin 454 

Malarial pigment 454 

Estimation of hemoglobin 454 

Direct methodsl 455 



XXVlll TABLE OF CONTENTS 

Page 

Indirect methods 455 

Hemometer of Fleischl-Miescher 456 

Hemoglobinometer of Dare 459 

Hemometer of Sahli 460 

Hemoglobinometer of Oliver 461 

Hemoglobinometer of Tallqvist 463 

Variations in amount of hemoglobin 464 

Oligo-chromemia 465 

Color index 465 

Proteins of the blood 466 

Other nitrogenous constituents 469 

Total nitrogen 469 

Total non-protein nitrogen 469 

Urea . 474 

Uric acid 481 

Ammonia 487 

Creatinin 490 

Amino-acids - 492 

Carbohydrates 493 

Pats and fatty acids . 498 

Acetone 502 

Biliary constituents 502 

Inorganic constituents 5^3 

Blood gases 508 

' Ferments of the blood 5^9 

Enumeration of the cells 5^9 

Hemocytometer of Thoma-Zeiss 510 

Hemocytometer of Durham 522 

Hemocytometer of Oliver 523 

III. Morphology of the Blood 524 

Examination of fresh blood 525 

Preparation of smears 526 

Fixation of smears 528 

Staining methods 531 

Erythrocytes 54 1 

Appearance and structure 54 1 

Size and shape 543 

Nucleation 545 

Number 54^ 

Normal variations . . - 548 

Pathological variations • • 55^ 

Oligo-cythemia 55^ 

Poly-cythemia 55 1 

Staining properties 552 

Degenerations 553 

Isotonicity and resistance 555 

Variations in childhood and old age 55^ 

Functions 557 

Leucocytes 557 

Appearance 557 

Types in normal blood 55^ 

Lymphocytes 55^ 

Large mononuclears 559~'^ 

Polymorphonuclear neutrophiles . 5^0 



TABLE OF CONTENTS XXIX 

Page 

Polymorphonuclear eosinophiles 562 

Polymorphonuclear basophiles 562 

Types in pathological blood 563 

Myelocytes 563 

Irritation forms 565 

Degeneration forms 565 

Differential counting 566 

Number 567 

Leacocytosis , . . 568 

Physiological . 569 

Pathological .^571 

Mixed leucocytosis ; . 574 

Lymphocytosis 574 

Eosinophilia 575 

Mast-cell type 577 

Leucopenia 577 

Variations in infancy and childhood 578 

Functions • . 578 

Blood-plates 580 

Appearance 580 

Size 580 

Number 580 

Staining properties .581 

Function ., 581 

Hemoconien 581 

JNIorphology of the blood-forming organs 582 

IV. P.A.THOLOGY OF THE Blood 584 

Special 584 

Anemia 584 

Primary 585 

Simple primary anemia 585 

Chlorosis 585 

Progressive pernicious anemia 587 

Splenic anemia 589 

Anemia infantum pseudo-leukemica 590 

Leukanemia . . . . : ', 590 

Aplastic anemia .......' 591 

Secondary 591 

Acute hemorrhage 592 

Chronic hemorrhage 593 

Inanition 593 

Intestinal parasites 594 

Fever 595 

Blood poisons 595 

Leukemia 596 

Spleno-myelogenous type 596 

Lymphatic type 599 

Acute type 600 

Pseudo-leukemia 601 

Hodgkin's disease 601 

Tuberculosis of the lymph glands 602 

Lympho-sarcoma 602 

Gummatous lymphoma 603 

General 603 



XXX TABLE OF CONTENTS 

Page 

' Blood changes following surgical intervention 603 

Constitutional diseases 604 

Diabetes mellitus 604 

Gout 605 

Addison's disease . 606 

Rickets 606 

Myxedema 606 

Acute infections 606 

Pneumonia 607 

Typhoid fever 609 

Scarlet fever 610 

Measles 611 

Variola 612 

Diphtheria 613 

Pertussis ■ 613 

Rheumatism 614 

Chronic infections 615 

Tuberculosis 615 

Syphilis 615 

Leprosy 616 

Carcinoma 616 

Effects of splenectomy 618 

V. Parasitology of the Blood 619 

Malaria 619 

Examination of fresh blood. 620 

Tertian parasite 620 

Quartan parasite 623 

Estivo-autumnal parasite 624 

Stained specimens 625 

Sporogony 629 

General hematological changes 630 

Relapsing fever . 631 

Sleeping sickness 632 

Kala-azar 634 

Filariasis 634 

Syphilis . 636 

Cultivation of treponema pallidum 639 

Yellow fever * 641 

Infectious jaundice 643 

Rocky mountain spotted fever 645 

Distomiasis 646 

VI. Bacteriology of the Blood 646 

Technic 647 

Organisms found in the blood 648 

VII. Serum Pathology 655 

Ehrlich's side-chain theory 656 

Phagocytosis 660 

Opsonins 660 

Allergic reactions 662 

Tuberculin reactions 663 

Luetin reactions 665 

Schick reaction '. . 667 

VIII. Sero-Diagnosis 669 

Agglutination reactions 669 



TABLE OF CONTENTS XXXI 

Page 

Gruber-Widal test 670 

Method of Bass and Watkins 674 

Diseases other than typhoid 676 

Precipitin reaction 677 

Complement-fixation test 680 

Wassermann reaction 683 

Noguchi method 693 

, Diseases other than syphilis 701 

Abderhalden's sero-diagnosis 703 

Of pregnancy 703 

Of other conditions 715 

Herman-Perutz reaction 717 

Coagulo Reaction 719 

Tests before transfusion 723 

IX. Medico-Legal Aspects 727 

Red cells ^ 727 

Guaiac test 728 

Schaer's test 729 

Phenolphthalin test 729 

Teichmann's test 731 

Spectroscopic examination 732 

Precipitin test 732 

X. Value and Limitations of Blood Examinations 732 

CHAPTER IX 

Transudates and Exudates 

I. General Considerations 736 

II. Physical and Chemical Properties 'j^'] 

Serous exudates 738 

Chylous exudates 739 

Chyloid exudates . . , 739 

Hemorrhagic exudates 739 

Purulent exudates 740 

Putrid exudates 740 

III. Bacteriology 741 

Tubercle bacilli 741 

Inoscopy 741 

Gonococci 741 

Smegma bacilli 743 

Ducrey's bacilli 744 

Spirochete pallidas 744 

IV. Cytology 748 

Technic 748 

Cytology of normal fluids 749 

Cytology of pathological fluids 749 

Pleural exudates 750 

Primary tubercular pleurisy 750 

Secondary tubercular pleurisy 750 

Pneumococcus pleurisy 750 

Streptococcus pleurisy 750 

Typhoid pleurisy 751 

Malignant pleurisy 751 



XXXll TABLE OF CONTENTS 

Page 

Nephritic and cardiac pleurisy 751 

Peritoneal exudates 751 

V. Cyst Fluids 752 

Ovarian cysts 752 

Serous cysts 752 

Myxoid or colloid cysts . 752 

Papillary cysts '. . . . 753 

Dermoid cysts 753 

Parovarian cysts 753 

Hydrocele ' . . 753 

Spermatocele 754 

Hydronephrosis 754 

Hydatid cysts 754 

Pancreatic cysts 754 

VI. Cerebrospinal Fluid 754 

Lumbar puncture 755 

Microscopic examination. 758 

Epidemic cerebrospinal meningitis 759 

Tubercular meningitis 760 

Acute anterior poliomyelitis 760 

Cerebrospinal syphilis .761 

Noguchi's butyric acid test , . 762 

Nonne's test 762 

Ross- Jones test 763 

Lange's colloidal-gold test 763 



CHAPTER X 

Secretion of the Mammary Glands 

I. General Considerations 766 

II. Physical and Chemical Properties 767 

Appearance and color 768 

Specific gravity 768 

Reaction 769 

Coagulation 769 

Total solids 769 

Ash 769 

Protein 770 

Total protein 770 

Method of Sebelien- 770 

Method of Boggs 770 

Casein 771 

Albumin and globulin 771 

Fat 772 

Babcock's method 772 

Extraction method . -, . . 773 

Lactose . 773 

Preservatives in cow's milk 774 

Sodium carbonate 774 

Salicylic acid 775 

Formaldehyde , . 775 

Boric acid and borax ., 775 

III. Bacteriological Examination of Milk 775 



TABLE OF CONTENTS XXXIU 

CHAPTER XI 
Clinical Bacteriology 

Page 

I. General Considerations yyB 

II. Sterilization 779 

III. Preparation of Culture Media 780 

IV. Incubation 784 

V. Preparation of Cultures 785 

VI. Staining 787 

VII. Identification of Organisms 789 

Diphtheria Bacillus 789 

Influenza Bacillus 791 

Pertussis Bacillus 791 

Typhoid Bacillus 792 

Colon Bacillus . 793 

Dysentery Bacillus 795 

Gas Bacillus 797 

Cholera Spirillum 799 

Pneumococcus 801 

Streptococcus 802 

Staphylococcus 804 

Gonococcus 805 

Meningococcus 806 

Micrococcus Catarrhalis , 807 

VIII Vaccines 808 

Preparation of vaccines 809 

Stock vaccines 809 

Mixed vaccines 810 

Autogenous vaccines 811 

Anti-typhoid vaccination 811 

Index. . . • / 815 



LIST OF ILLUSTRATIONS 



PLATES 

To Face Page 

I. Tubercle Bacilli in Sputum 20 

II. Streptococcus Pyogenes 26 

III. Leptothrix and Spirocheta Buccalis (Unstained) ...... 35 

IV. Diphtheria BacilU Showing Polar Staining 38 

V. Endamebae Gingivalis (Gros.) . 42 

VI. Koch- Weeks Bacillus .48 

Morax-Axenfeld Diplobacillus 48 

VII. Trachoma Bodies of Prowazek-Greeff 49 

VIII. Vegetable Cells found in Feces ..;.-. .158 

IX. Osazons . '. 310 / 

X. Ammonium Urate Crystals 365 

XI. Waxy Casts 377 

XII. Mucous Threads in Urine (Unstained) 379:. 

XIII. Cystitis Due to Colon Bacillus 384 .-. 

XIV. Staphylococcus Cystitis 385 .,. 

XV. Absorption Spectra 450 ^ 

XVI. Absorption Spectra 451 

XVII. Fresh Normal Blood . . 525 

XVIII. Types of Red Cells 544 

XIX. Ring Bodies in Red Cells 554 

XX. The Leucocytes 558 

XXL lodophilia 563 

XXII. Polynuclear Leucocytosis 568 

XXIII. Chlorotic Anemia 586 

XXIV. Blood in Pernicious Anemia 588 

XXV. Blood in Leukanemia 590 

XXVI. Blood in Spleno-myelogenous Leukemia 597 

XXVII. Lymphatic Leukemia 600 

XXVIII. The Tertian Parasite (Unstained) 621 

XXIX. The Quartan Parasite (Unstained) 623 

XXX. The Estivo-autumnal Parasite (Unstained) 624 

XXXL Tertian Parasite (Stained) 626 

XXXII. Estivo-autumnal Parasite (Stained) 627 

XXXIII. Gonococci in Urethral Discharge 742 

XXXIV. Spirochetae Pallidae in Tissue . 746 

XXXV 



XXXVl LIST OF ILLUSTRATIONS 

To Face Page 

XXXV. Exudate from Tubercular Pleurisy 750r 

XXXVI. Exudate in Pneumonic Pleurisy 75 1 

XXXVII. Exudate in Malignant Pleurisy 752 , 



FIGURES 



Page 

1. Curschmann's Spirals 7 

2. Objects Found in the Sputum 10 

3. Aspergillus Fumigatus 15 

4. Micrococcus Catarrhalis 15 

5. Budding Forms of Blastomycetes. 16 

6. Diplococcus Pneumoniae 25 

7. Friedlander's Bacillus 26"^ 

8. Bacillus Influenzae ... 27 

9. Actinomyces . 28 

10. Paragonimus Westermanii . 29 

11. Ovum of Paragonimus Westermanii 30 

12. Vincent's Spirillum and Bacillus 40 

13. Oidium Albicans 41 

14. Stomach Tube 52 

15. Turck's Aspiration Apparatus 53 

16. Boas-Oppler Bacillus 63 

17. Strauss' Separatory Funnel . 76 

18. SahH's Desmoid Bag 88 

19. Normal Feces 100 

20. Boas' Stool-Sieve 104 

21. Schmidt's Fermentation Apparatus 124 

22. Cholera Spirilla 129 

23. Bacillus Typhosus 130 

24. Amoeba Coli 136 

25. Coccidium Hominis ' 137 

26. Trichomonas Intestinalis 140 

27. Cercomonas Hominis •. 140 

28. Megastoma Entericum 141 

29. Balantidium Coli 142 

30. Taenia Solium 143 

31. Taenia Saginata : 144 

32. Taenia Cucumerina 144 

2,2,- Taenia Nana 144 

34. Taenia Diminuta 145 

35. Ovum of Taenia Diminuta 145 

36. Taenia Echinococcus 146 



LIST OF ILLUSTRATIONS XXXVIl 

Page 

37. Hydatid Cyst 146 

38. Bothriocephalus Latus 147 

39. Dibothriocephalus Cordatus 147 

40. Ascaris Lumbricoides 148 

41. Ascaris Mystax 149 

42. Oxyuris Vermicularis 150 

43. Strongyloides Intestinalis 151 

44. Trichiuris Trichura 152 

45. Trichinella Spiralis 153 

46. Tail, of Uncinaria Duodenalis 154 

47. Anterior End of Uncinaria Duodenalis : 154 

48. Tail of Uncinaria Americana 156 

49. Anterior End of Uncinaria Americana 156 

50. Parasitic Bodies, Ova, and Larvae 157 

51. Fasciola Hepatica . 160 

52. Fasciolopsis Buski 161 

53. Opisthorchis Felineus 162 

54. Opisthorchis Sinensis 163 

55. Eustrongylus Gigas 164 

56. Acarus Scabiei 165 

57. Demodex Folliculorum 166 

58. Leptus Autumnalis 166 

59. Pediculus Capitis. . . _ 167 

60. Pediculus Vestimenti 167 

61. Pediculus Pubis ' 167 

62. Pulex Irritans 168 

63. Pulex Penetrans 169 

64. Achorion Schonleinii . 170 

65. Normal Hair 171 

66. Trichophyton Endo-ectothrix 171 

67. Microsporon Audouini 172 

68. Mycelial Threads of Blastomycetes 173 

69. Budding Forms of Sporothrix Schenckii . • i74 

70. Urinometer and Cylinder 193 

71. Volumetric Flasks 208 

72. Kjeldahl's Nitrogen Apparatus 22^, 

73. Doremus' Ureometer 231 

74. Doremus-Hinds Ureometer 231 

75. Folin's Urea Apparatus ... 27,2 

76. Schlosing's Ammonia Apparatus 239 

77. Folin's Ammonia Apparatus 240 

78. Folin's Absorption Bulb 241 

79. Ruhemann's Uricometer 251 

80. Sargent's Colorimeter 257 

81. Conical Test-glass 2St, 

82. Horismascope 2^2> 



XXXVUl LIST OF ILLUSTRATIONS 

■ [ . 

Page 

83. Esbach's Albuminometer 287 

84. Laurent Polariscope 317 

85. Diagrammatic Representation of the Course of Light through the 

Laurent Polariscope 318 

86. Einhorn's Saccharometer 319 

87. Lohnstein's Fermentation Tube for Undiluted Urine 321 

88. Lohnstein's Fermentation Tube for Diluted Urine 321 

89. Purdy Electric Centrifuge 354 

90. Sediment Tube 355 

91. Percentage Centrifuge Tube 355 

92. Various Forms of Uric Acid 357 

93. Acid Sodium Urate 358 

94. Xanthin 358 

95. Calcium Oxalate 359 

96. Cystin 360 

97. Pure Leucin 361 

98. Impure Leucin 362 

99. Tyrosin 362 

100. Calcium Sulphate . 363 

loi. Bilirubin 363 

102. Cholesterin . 365 

103. Magnesium- Ammonium Phosphate 366 

104. Calcium Carbonate 367 

105. Urinary Epithelium 368 

106. Pus Corpuscles 370 

107. Hyaline Casts 374 

108. Granular Casts . . . . ; 376 

109. Epithelial Casts 377 

no. Fatty Casts 378 

111. Blood, Pus, Hyaline, and Epithelial Casts 379 

112. Cylindroids . 380 

113. Scolex and Hooklets of Taenia Echinococcus in Urine 384 

114. Ova and Miracidium of Schistosomum Hematobium 385 

115. Normal Semen 399 

116. Chorionic VilU 406 

117. Daland's Hematocrit : 413 

118. Hematocrit Tube 414 

119. Blood Needle 416 

120. Pycnometer f 439 

121. Boggs' Coagulometer 443 

122. Beckmann Apparatus 444 

123. Direct-vision Spectroscope 449 

124. Hemometer of Fleischl-Miescher 45^ 

125. Hemoglobinometer of Dare 459 

126. Method of Filling the Dare Blood Pipet 460 



LIST OF ILLUSTRATIONS XXXIX 

Page 

127. Hemometer of Sahli 461 

128. Hemoglobinometer of Oliver 462 

129. Tallqvist's Hemoglobinometer 463 

130. Thoma-Zeiss Counting Chamber 510 

131. Diluting Pipets 511 

132. Ruled Surface of Thoma-Zeiss Counting Chamber 512 

133. Turk's Ruling of Counting Chamber 513 

134. Plan of Counting the Cells 519 

135. Cross-section of Durham's Blood Pipet 522 

136. Oliver's Hemocytometer 523 

137. Preparation of Blood-smears with Glass Slides 526 

138. Preparation of Blood-smears with Cigarette Paper 527 

139. Ehrlich Forceps 527 

140. Pinch Forceps 528 

141. Oven for Fixing Blood-films 529 

142. Normal Blood Showing Rouleaux Formation and Fibrin Network . 541 

143. Cycles of the Malarial Parasite 628 

144. Spirillum of Obermeier . 632 

145. Trypanosoma Gambiense 633 

146. Filaria Bancrofti 635 

147. Spirochetae Pallidas and Refringens 6^8 

148. Ultra-condenser of Reichert 639 

149. Schistosomum Hematobium 645 

150. Illustrating the Mechanism of Toxin-cell Union ........ 656 

151. Illustrating the Elaboration and Action of Antitoxin 657 

152. Illustrating the Mechanism of Hemolysis 658 

153. Illustrating the Mechanism of Antihemolysis 659 

154. Bacillus Typhosus at Beginning of Widal Test ......... 669 

155. A Pseudo-Widal Reaction 670 

156. A Positive Widal Reaction 670 

157. Hemin Crystals from Human Blood 730 

158. Lumbar Puncture 756 

159. Diplococcus Intracellularis Meningitidis 759 

160. Normal Milk and Colostrum .- . 766 

161. Babcock Milk and Cream Bottles 772 

162. Bottle for Human Milk 772 

163. Soxhlet Apparatus 774 

164. Wolfhiigel's Colony Counter 776 

165. Lautenschlager Hot Air Sterilizer 778 

166. Autoclave 779 

167. Arnold Sterilizer 780 

168. Incubator 785 

169. Platinum Needles 786 

170. Apparatus for Anaerobic Cultivation 787 



DIAGNOSTIC METHODS 



CHAPTER I 
THE SPUTUM 

I. General Considerations 

At the present time the examination of the sputum is more or less lim- 
ited to the search for various specific organisms, especially the bacilli of tuber- 
culosis and of pneumonia. This is very much to be regretted, as frequently 
the appearance, amount, consistency, color, and other characteristics are of 
great aid and have led our older brothers to correct diagnoses before the days 
of microscopic examination.^ 

The sputum, strictly speaking, should be considered as the material which 
comes from the respiratory passage anywhere along its course. It may be, 
therefore, of laryngeal, bronchial, or alveolar origin. More commonly, how- 
ever, we find the sputum considered by the general practitioner and by the 
laity as anything which is expectorated, so that specimens. which consist of 
nothing more than salivary secretion are frequently sent to laboratories for 
examination. In some cases, especially of inflammation of the naso-pharynx 
or perforation from neighboring organs, this buccal secretion may be mixed 
with material from the nose, mouth, ear, or esophagus. For these reasons, 
if for no others, it is absolutely essential that frequent examinations of 
sputum, in the strict sense, be made before a negative diagnosis of a suspected 
condition may be given. 

As a rule, any sputum at all should be considered pathological, as normal 
persons raise httle or nothing from the lungs at any time. By this is not 
meant that serious disease may obtain when a small amount of sputum exists, 
as patients suffering with catarrhal conditions of the naso-pharynx frequently 
have an accumulation of material, which has settled in the bronchial tubes 
over night and is raised in the morning. Those of us who are unfortunate 
enough to live in atmospheres which are loaded with soot and dirt frequently 
raise a certain amount of sputum which arises from an increased activity of 
the mucous membrane of the respiratory passages to compensate for the 
dryness and irritation which these foreign substances have caused. This 
morning sputum is small in amount and is in the form of large, tough, 
elastic masses which very much resemble boiled sago. These masses are 
at times extremely dark in color, due to the dirt which has been taken in 
with the inhaled air. In such sputum we find much mucus, degenerated 
epithelium, pus cells and various micro-organisms. These organisms are 

^See Laird, Jour. Am. Med. Assn., 1915, LXIV, 427, 



2 DIAGNOSTIC METHODS 

rarely of pathologic significance, although many pathogenic types may 
be present. 

The sputum should be collected in receptacles which may be completely 
and easily disinfected. The best receivers for the sputum are the ordinary 
paper spit-cups which can be burned as occasion may demand. The practice 
of expectorating upon cloths is only to be advised when these cloths are imme- 
diately burned. In the spit-cup should always be contained a certain amount 
of disinfectant, such as dilute carbolic acid or corrosive sublimate solution, 
so that no chance of transference of infection may arise. Neglect of such 
precautions has frequently led to serious consequences in the case of the 
healthy members of the family. When sputum is to be collected for exami- 
nation, the material raised by coughing should be received in a wide-mouthed 
bottle which contains no disinfectants, as these agents coagulate the protein 
material of the infecting organism and in some cases change its staining 
characteristics. Immediately after examination of such material it should, 
of course, be treated with the disinfecting solution. 

II. Physical and Chemical Characteristics 

Amount. 

Some general idea of the amount of sputum expectorated is always advis- 
able. It is rarely necessary, however, to make any collection of the material 
during the 24 hours' period, as one can usually gain the information by ques- 
tioning the patient or nurse as to the amount passed from time to time. The 
amount of sputum expectorated in 24 hours depends very much upon the 
nature of the pathologic condition. In cases of so-called dry bronchitis, dif- 
fuse bronchitis, early tuberculosis, and occasionally of lobar pneumonia, the 
sputum is so viscid that there is practically none obtained. In cases of chronic 
bronchitis, tuberculosis with cavity formation, and bronchiectasis we find 
large amounts; while in cases of lung abscess or of perforating pleurisy, blood 
or pus may flow from the mouth in very large quantities. The absolute 
amount of sputum may, therefore, vary from a cubic centimeter to a Kter 
or more. An extensive expectoration will naturally have more or less serious 
effect upon the patient's general nutrition, so that we are not surprised to 
find cases in which as high as 5 per cent, of the total nitrogen eliminated 
passes from the system in the sputum (Lenz). 
Consistency. 

As a rule, the consistency of the sputum varies from that of a liquid to a 
highly tenacious material, inversely as the amount of sputum. This latter 
Statement holds in most cases with the expection of pneumonia, in which we 
have an extremely tenacious sputum and, also, a very abundant one. Just 
what substance induces this extreme tenacity is uncertain, but it may be gener- 
ally said that mucin is the causative factor, although the tenacious pneumonic 
sputum shows very little mucin. According to Kossel, the tenacity may be 
due to the presence of nuclein derivatives. In the early stages of acute bron- 
chitis, or bronchial asthma, and in whooping-cough the sputum is usually very 



THE SPUTUM 3 

tenacious and ropy; while in edema of the lungs, pulmonary abscess, putrid 
bronchitis, and pulmonary gangrene it is very watery and contains large 
numbers of pus-cells. 

Reaction. 

The fresh specimen of sputum is usually alkaline in reaction. However, 
in cases in which the sputum has remained in the lungs for some time, as in 
cavity formation, the reaction is acid. 

Color. 

The color of the sputum may range from that of a colorless material to one 
showing any of the tints of the rainbow. These colors are due to admixtures 
of various abnormal products with the sputum. A bright red sputum is sig- 
nificant of the presence of blood, according to Traube, unchanged red blood- 
cells necessarily being present. The amount of blood may vary from a light 
streaking of the sputum to one showing a deep red, rusty, or prune-juice color. 
Such bloody sputa are found after trauma, pneumonia, gangrene, hemorrhagic 
infarction of the lungs, chronic passive congestion, as well as adventitious mix- 
ture of sputum with nasal or pharyngeal material containing blood. The 
blood may be due to rupture of a vessel and may then constitute the condition 
known as pulmonary hemorrhage. This condition of hemoptysis^ is fre- 
quently confounded with that of hematemesis and is differentiated by the 
fact that the blood in hemoptysis is frothy, bright red in color, alkaline in 
reaction, and usually associated with mucopurulent material ; while the blood 
in hematemesis is frequently dark and grumous, usually clotted and acid in 
reaction. In sputa which are tinted by changed hemoglobin, the color is most 
varied. Many oxidation products of hemoglobin are found in the sputum, 
depending upon the time which the sputum has lain in the lungs. Thus, for 
instance, in pneumonia we find a rusty, prune-juice colored sputum, whose 
color seems to be due to an unknown derivative of hemoglobin. The shade 
of color in pneumonia may, however, range from red through brown to green. 
In cases of mitral disease associated with passive congestion of the lungs we 
find, frequently, a light brown color, due to the presence of hematoidin gran- 
ules in the epithelial cells. 

In many cases of abscess of the liver which perforates into the lung or 
in catarrhal jaundice, bile pigments may be found in the sputum which may 
give rise to various colored sputa from red through blue to green. Although, 
as shown in a later table, the bile pigments and hemoglobin are very closely 
related chemically, yet the clinical significance of the appearance of bile pig- 
ment in such cases as the above is very important. In many cases of true 
jaundice the sputum, in case any exists, may show a distinct grass-green 
color due to the presence of oxidized bile pigments. This same green color 
frequently appears in the sputum, however, in cases of croupous pneumonia 
during lysis. In this latter case the color is due, probably, to the same pig- 
ment which is oxidized before expectoration. In these cases of pneumonia 
with green sputum and no jaundice, a fresh involvement of the lung is usually 

^ See van der Hoeven-Leonhard, Arch, internat. de laryngoL, 1913, XXXV, 831. 



4 DIAGNOSTIC METHODS 

associated with a rusty sputum. Besides jaundice and pneumonia we may 
have, as causes of green sputum, certain chloromata of the lungs as well as the 
development of chromogenic bacteria within the lungs. This action of 
chromogenic bacteria is not always observed when the sputum is expectorated, 
but may appear only after the sputum has stood. The presence of the bacillus 
pyocyaneus may give a sputum which is brilHant blue or greenish in tint. 
Sputa very frequently show changes in color as well as consistency due 
to various substances inhaled. Thus we find a distinctly black sputum, in 
cases of anthracosis, in coal-miners and in many city residents. So frequent 
is this occurrence that the lung tissue may even be invaded by the coal pig- 
ment.^ Workers in bronze and brass as well as other metals frequently show 
a sputum tinged red with ferric oxide, arising from a condition of the lung 
known as siderosis.^ Stone-cutters frequently show much stone-dust in the 
sputum which is characteristic of the condition of chalicosis, stone-cutters' 
phthisis, or grinders ' rot. Workers in flour mills and in bakeries frequently 
expectorate doughy masses, while those in cottom mills show the presence 
of cotton fibers in their sputum. Finally it may be said that the color of the 
sputum may be changed by the presence of certain foods, such as milk, eggs, 
and chocolate, while tobacco users frequently have a sputum tinged dark 
brown. 

Odor. 

Ordinarily the sputum has no odor unless it has stagnated either in the 
receiving cup or in the system. Such old sputum sometimes has a very dis- 
tinct putrefactive odor. The odor of sputum in tuberculosis and bronchiec- 
tasis is peculiarly heavy and sweet, while that in putrid bronchitis is often 
extremely offensive. In cases of perforating empyema a peculiar cheese-like 
odor is observed. While these odors are not characteristic in themselves, 
they are usually more or less significant of the condition with which they are 
commonly associated. 

Character of the Sputum. 

The character of the sputum has reference more to the apparent com- 
position of the sputum than to its consistency. Air is usually present in the 
sputum in various amounts, so that one may judge, from the size of the air- 
bubbles, of the size of the bronchi from which the sputum came. The sputum 
from cavities and large bronchi contains no air and, therefore, sinks in water. 
This is the so-called sputum fundum petens. 

Sputum known as mucoid sputum is glairy, transparent, and tenacious, 
becoming cloudy on the addition of acetic acid due to the precipitation of 
mucin. This type of sputum is found particularly in acute bronchitis and 
in asthma. 

A mucopurulent sputum is one containing both pus and mucoid material. 

Small amounts of pus give a whitish color, either to the whole or to portions of 

the sputum, the pus being observed in masses or in streaks through the 

mucoid material. Larger quantities of pus give a yellowish or occasionally a 

^ See Filadoro, Med. nuova, 1913, IV,i33; also, Haythorn, Jour. Med. Research, 1913, 
XXIX,259; Klotz, Am. Jour. Pub. Health, 1914, IV, 887. 

2 See Gigon, Beitr. z. path. anat. u. z. allg. Path., 191 2, LV, 46. 



THE SPUTUM 5 

yellowish green tinge to the sputum. In this latter type the pus and mucus 
seem to be mixed homogeneously. In the sputum from cavities we find the 
mucopurulent material arranging itself flatly like a coin, constituting the so- 
called '^ nummular^' sputum. 

Purulent sputum is found in cases of ruptured empyema, abscess of the 
lung, and in some cases of bronchiectasis. This purulent sputum differs from 
the mucopurulent type in the fact that the pus is much more abundant and is 
almost in the pure state, being mixed with a small amount of tenacious mucus. 

In some cases, especially in edema of the lungs, a sputum is obtained, 
known as serous sputum, which is colorless and quite frothy. This sputum 
resembles very closely the ordinary salivary secretions and should not be 
confused with it. 

In many cases, especially in putrid bronchitis, gangrene of the lung, and 
bronchiectasis, the sputum on being voided into a cylinder will separate into 
three distinct layers, occasionally into four. The upper layer is of frothy 
mucus; a second layer, which is not always present, consists of certain albu- 
minous material which hangs in long shreds down into the third layer, which 
consists of the sero-pus and is usually opaque and watery. The bottom layer 
contains the morphological elements, pus, tissue shreds, and bacteria. 

Besides the varieties of sputum named above, the admixture of blood may 
give rise to sputum which is known as sanguinous sputum or as sanguino- 
mucopurulent or purulent sputum. 

Chemical Properties. 

The chief chemical examination is applied to the detection and estimation 
of the amount of albumin and of mucin in the sputum. The method is as 
follows:^ Treat the sputum with an equal quantity of 3 per cent, acetic acid, 
in order to precipitate the mucin. Shake thoroughly for a few minutes and 
allow to stand for 15 minutes. Filter through filter-paper until a perfectly 
clear filtrate is obtained, adding a known amount of water to facilitate 
filtration if necessary. Apply to this filtrate any of the tests for albumin 
given in the section on urine. If the quantitative determination is desired, 
use Tsuchiya's reagent as the precipitant, multiplying the result by the di- 
lution of the sputum to obtain the true percentage of albumin. 

A large amount of albumin indicates, of course, such conditions as pneu- 
monia, pulmonary edema or perforating empyema. While the early workers 
regarded anything more than a slight opalescence as pathological, a trace of 
albumin indicating a pulmonary tuberculosis in their opinion, later investiga- 
tors showed that albumin is present in the sputum whenever there is active 
exudation into the alveoli as well as into and under the pleura. Many more 
recent studies have proven that albumin may not be present in the sputum of 
true pulmonary tuberculosis, but is frequently seen in benign affections and 
when present is due, in the majority of cases, to occult blood. For these 
reasons it is more than probable that such chemical tests are to be given 
merely a presumptive interpretation. ^ 

^Lesieur and Prires, Paris med., 1911, IV, 29. 

2 See Waniier, Deutsch. Arch. f. klin. Med., 1903, LXXV, 347; Roger and Levy- Valensi, 
Presse med., 1910, XVIII, 289; Ibid., 1911, XIX, 409; Geeraerd, Jour, de med., de chir. et 



6 DIAGNOSTIC METHODS 

Besides these albuminous principles, the sputum, especially in cases of 
gangrene and putrid bronchitis, contains a ferment very much resembling in 
its action the trypsin of the pancreatic juice (Stolnikow) . ^ This ferment seems 
to indicate a highly destructive process in the lung tissue. Other chemical, 
substances, such as glycogen and fatty acids, ^ are frequently found in the 
sputum but may be passed with mere mention. The so-called myelin granules 
or globules, which appear in the alveolar cells of the sputum, consist largely of 
fatty principles, such as protagon, lecithin and cholestrin. 

III. Macroscopic Examination 

While much that is included in the previous section would come properly 
under the head of the macroscopic examination of the sputum, the writer has 
reference more, in this connection, to the appearance of macroscopic elements 
as distinguished from those which are purely microscopic. 

(i) Cheesy Masses. 

Frequently one finds in the sputum small cheese-like particles which 
vary in size from that of a pin-point to that of a pea, the large majority being 
about the limit of ordinary vision. These cheesy masses are fragments of 
necrotic tissue and appear in the larger form in cases of abscess or gangrene of the 
lung, while in tuberculosis they are always small unless the cavity, from which 
the material is derived, is markedly necrotic. The color of these masses varies 
from a yellow to a black. Those fragments which come from an abscess are of 
yellow color due to the presence of much pus, the darker ones contain decom- 
position products of hemoglobin, while many of them are tinged a deep black 
with coal pigment. If the sputum be squeezed between two glass plates 
these cheesy particles or fragments can sometimes be more distinctly seen. 
They are present in largest numbers in the so-called "nummular" sputum 
from a tuberculous cavity. 

(2) Dittrich's Plugs. 

These masses are similar to the small caseous particles above mentioned 
and are frequently expectorated by perfectly normal individuals. The true 

de Pharmacol., 1910, XV, 505; Raymond, Presse med., 1911, XIX, 675; Goodman, Arch., 
Int. Med., 1911, VIII, 163; Fishberg and Felberbaum, Med. Record, iqii,LXXX, 870; Fish- 
berg, i\rch. Diagnosis, 1912, V, 220; Works, Jour. Am. Med. Assn., 1912, LIX, 1537; Scott, 
Ibid., i9i3,LX, 440; Acs-Nagy, Wien. klin. Wchnschr., 191 2, XXV, 1904; Kauffmann, Beitr. 
z. Klin. d. Tuberk., 1913, XXVI, 269; Schmitz, Med. Klin., 1913, IX, 1163; Berkovits 
and Rudas, Berl. klin. Wchnschr., 1913, L, 1752; Ridge and Treadgold, Lancet, 1913, II, 
382; Schneider, Zentralbl. f. inn. Med., 1913, XXXIV, 1025; Gelderblom, Deutsch. med. 
Wchnschr., 1913, XXXIX, 1987; Maliwa, Deutsch. Arch. f. klin. Med., 1913, CXII, 231; 
Hohn and Himmelberger, Jour. Am. Med. Assn., 1914, LXII, 20; Lewis, New York Med. 
Jour., 1914, XCIX, 1031; Melikjanz, Med. Obozr., 1914, LXXX, 819; Beitr. z. Klin. d. 
Tuberk., i9i4,XXX, 81; Wien. klin. Wchnschr., 1914, XXVII, 653; Cocke, Am. Jour. Med. 
Sc, 1914, CXLVIII, 724; Hafemann, Deutsch. med. Wchnschr., 1914, XL, 1715; Benzler, 
Beitr. z. Klin. d. Tuberk., 1914, XXXII, 363; Glover, Brit. Jour. Tuberc, i9i4,VIII, 217; 
Holm and Chambers, Jour. Michigan Med. Soc, 1914, XIII, 226; Callender, Med. Record, 

1914, LXXXVI, 783; Fantoni, Gazz. d. osp., 1914, XXXV, 417; Lowenbein, Ztschr. f. 
Tuberk., 1915, XXIII, 122; Szaboky, Ibid., 352; Snijders, Nederl. Tijdschr. v. Geneesk., 

1915, I, 349; Lockwood, Jour. Am. Med. Assn., 1915, LXIV, 574. Holm and Chambers, 
Jour. Lab. and Clin. Med., 1916, 1, 519, Salomon, Presse Med., 1919, XXVII, 523. 

^See Eiselt, Ztschr. f. klin. Med. 191 2, LXXV, 71; also, MaHwa, Deutsch. Arch. f. 
klin. Med., 1914, CXV, 407. 

2 See Barbaro, Gazz. d. osp., 1915, XXXV, 985. Cox, Journal-Lancet, 1916, XXXVI, 
515- 



THE SPUTUM 7 

plugs are distinct casts of the bronchi or bronchioles and vary in size from 
pin-point to that of a bean, the majority being about the size of a mustard 
seed. The smaller ones are opaque and yellowish-white in color, while the 
larger ones have a distinct gray tinge. They are usually expectorated free 
from pus or mucus, so that they frequently give rise to anxiety, especially in 
those of a hypochondriac tendency. These plugs have a distinctly disagree- 
able odor, which is more evident if they are crushed on the glass plates. 
Microscopic examination of these masses shows large clumps of bacteria, fatty 
acid crystals, free fat globules, and cellular detritus. Occasionally a few 
leucocytes are found, but these are rare, while pigment granules, either of 
hematogenous or extraneous origin, are sometimes observed. While such 
plugs are especially numerous in cases of putrid bronchitis and bronchiectasis, 
they are frequently found in the crypts of the normal tonsil as well as in cases 
of follicular tonsillitis or of ozena. 






k^ 



I. TT. 

Fig. I. — Curschmann's spirals (Tyson after Curschmann). 
II and III, enlarged; a, a, central thread. 



nr. 

I, Natural size; 



(3) Curschmann's Spirals. 

These structures are found in the sputum in practically every case of 
true bronchial asthma and have been reported in acute bronchitis, croupous 
pneumonia, chronic pulmonary tuberculosis, and occasionally in chronic 
bronchitis. They are not present in every paroxysm of asthma, but are more 
frequently found just at the end of the paroxysm and are absent when the spu- 
tum becomes mucopurulent. They seem to be derived through true exuda- 
tion fro4ii the bronchioles, as Curschmann says, a bronchiolitis exudativa. 
These structures are recognizable, to a certain extent, by the naked eye, but 
for their absolute identification a microscopic examination is essential. They 
are composed of a spirally-twisted network of very delicate fibrils, in the 
meshes of which are numerous epithelial cells and eosinophile leucocytes. 
Along with these cellular bodies one frequently finds large numbers of diamond- 
shaped crystals, known as the Charcot-Leyden crystals, which will be dis- 
cussed later. This spirally-twisted mass (the mantle) seems to be wound 



8 DIAGNOSTIC METHODS 

around a central light thread. While this is the structure of the complete 
spiral, we frequently find variations pointing apparently to two distinct forms. 
The first is the spirally-twisted strand of mucus with the enclosures above 
mentioned. The second is the tight spiral mass of mucus wound around a 
central fibre. This central fibre is very refractive and is homogeneous in 
structure, varying in size from J^ to i8 microns in diameter. The length 
of these spirals is from i to 2 cm. and their breadth about i mm. 

(4) Fibrinous Casts. 

By the term fibrinous cast we refer more directly to true bronchial casts,, 
which are composed of fibrin. These are observed in pneumonia, in which case 
they are derived from the smaller bronchioles and are brownish or reddish in 
color and contain many red and white blood-cells. These smaller casts vary 
from 3^ to 3 cm. in length. In the chronic fibrinous bronchitis we find the so- 
called arborescent casts which are usually whitish in color and contain many 
epithelial cells. These casts vary in size from i to 15 cm. in length by several 
mm. in thickness. We may have an acute form of fibrinous bronchitis accom- 
panying various febrile conditions, so that similar casts may appear in almost 
any of the infectious diseases. These larger casts are fairly firm, usually 
have a lumen, and branch dichotomously five to ten times. Microscopic ex- 
amination shows them to consist of large numbers of longitudinal fibers con- 
taining blood and epithelial cells in their meshes. They may be stained with 
the Weigert fibrin stain in a very beautiful way. Such staining methods 
show that not all the material in such casts is fibrin, so that it may be neces- 
sary to rename these structures as simple bronchial casts rather than as 
fibrinous forms. 

Besides' these fibrinous casts, one occasionally may find distinct bronchial 
casts composed of the mycelium of various fungi. Thus mycelial threads of 
the aspergillus have been reported by Osier, Devillers and Renon. 

(5) Concretions. 

This name is applied to anything, expectorated with the sputum, which 
has the appearance or consistency of a stone. These concretions are formed 
in dilated portions of the bronchi or in cavities by the calcification of the stag- 
nated contents. Although concretions consisting of cartilaginous or osseous 
material are frequently found postmortem, yet there seems to be little men- 
tion in the hterature of any such formations being expectorated.^ 

{a) Bronchioliths. 

These concretions are formed by the deposition of calcium salts in the 
stagnated contents of the bronchus or of a cavity. They may be derived from 
the smaller or larger bronchi, but rarely form arborescent shapes, being usu- 
ally irregular and varying in size from a pin-head to that of a walnut. They are 
usually single, but may be multiple. They vary in consistency from that of 
chalk to a stone, and may be expectorated in small numbers over long periods 
of time. 

^ See Cade and Guerin,Lyonmed., 1 914, XL VI, 221. 



THE SPUTUM 9 

(b) Pneumoliths. 

These lung stones are in the majority of cases of tuberculous origin. 
They usually arise by the calcification of caseous areas which later ulcerate 
into a bronchus and are expectorated unless too large. They may, also, arise 
by the calcification of a pulmonary cavity or of a bronchial lymph-gland. 
These lung stones consist of the carbonates, phosphates, and sulphates of cal- 
cium and magnesium, in some one or more salts predominating, while in others 
still different combinations may exist. These pneumoliths either have a 
chalky or a calcareous consistency and vary in size from that of a pin-head to 
that of a tennis-ball. These lung stones are usually expectorated en masse 
or in the form of smaller portions of a larger stone. In some cases these 
smaller stones may reach the number of 500 (Portal). 

(6) Echinococcus Membranes. 

Rarely one may find in the sputum fragments of the walls of echinococcus 
cysts or their contents. These may come from a perforating cyst of the liver, 
kidney, or lung. The presence of the laminated membrane and of the parasitic 
scolices and hooks makes it possible to arrive at an absolute diagnosis of the 
origin of such material. The membrane is thick, tough, and of a porcelain- 
like color and may show a laminated or fibrillated structure. The parasite is 
discussed in a later section. 

(7) Foreign Bodies. 

Examination of the sputum may reveal the presence of material which 
has lodged in the air-passages and been retained for long periods of time. 
Such bodies are coins, fish-bones, and cherry-stones. Heyfelder reports a 
case of the expectoration of a wooden cigar-holder iij^ years after its 
disappearance. 

IV. Microscopic Examination 

The microscopic examination of the sputum is almost the only one to 
which it is subjected at the present day. This is to be regretted, as much may 
be learned from a careful macroscopic examination. However, a microscopic 
examination reveals evidence which points to an absolute diagnosis more fre- 
quently than does the macroscopic examination. Before making the micro- 
scopic examination, it is wise to place the sputum in a flat-bottomed dish 
(Petri dish) which has half of its base blackened, so that the more suspicious 
particles may be selected for microscopic investigation. Some experience in 
this work is necessary, as one is frequently called upon to recognize material 
which is purely extraneous and has absolutely nothing to do with the sputum. 
Such material practically always comes from the buccal cavity and consists of 
fragments of various food-stuffs, such as bread, fruit pulp, meat fibers, vege- 
table tissue, and portions of tobacco leaf. Naturally, such material should 
not mislead one, but it very frequently does. The fragments of meat tissue 
contain elastic tissue fibers and may lead one to state that such material is 
present in the sputum, thus giving expression to the possibihty of a diagnosis 
of incipient tuberculosis. 

A portion of the sputum selected for examination is taken up with a plati- 



lO 



DIAGNOSTIC METHODS 



num loop and spread in a thin layer upon a glass slide. It is then dried by 
passing the slide several times through the flame, care being taken not to burn 
the specimen. The smear is allowed to cool and is then stained either with 
Loflfler's methylene blue for general purposes or with special stains for the 
various specific organisms. 
(a) Pus-cells (Leucocytes). 

There is practically no specimen of sputum which does not contain leuco- 
cytes in larger or in smaller numbers. The true pus-cell is the polymorpho- 
nuclear neutrophile and appears in the sputum frequently filled with fat 
globules or pigment granules. In cases of asthma, the eosinophile cells are 
very abundant, while basophile cells may occasionally obtain. Although 




Fig. 2. — Objects found in the sputum (Landois). i, Detritus and dust-particles; 2, pig- 
mented alveolar epithelium; 3, fatty degenerated and partially pigmented alveolar epithe- 
lium; 4, alveolar epithelium showing myelin-degeneration; 5, free myelin forms; 6, 7, des- 
quamated cihated epithelium, partly changed and deprived of its cilia; 8, squamous epithe- 
lium from the mouth; 9, leucocytes; 10, elastic fibers; 11, fibrinous cast of a small bronchus; 
12, leptothrix buccalis, together with cocci, bacilli, and spirochete; a, fatty acid crystals 
and free fatty granules; 6, hematoidin; c, Charcot's crystals; d, cholesterin. 

these cells are so frequent in asthma, a diagnosis should not rest upon such 
evidence alone, as there seems to be a form of bronchitis, possibly of the tuber- 
cular variety, which has been named '^eosinophilic bronchitis" from the 
large number of such cells observed. In pulmonary tuberculosis, which is not 
associa,ted with a mixed infection, it is very common to find the small mono- 
nuclear leucocyte (lymphocyte) in place of the polynuclear type. This finding 
is of such frequent occurrence that the writer is often led to search many slides 
for tubercle bacilli in case the organisms have not been found in the earlier 
specimens examined. Basophilic cells are not infrequently observed in the 
sputum. Pottenger^ reports the presence of macro-phagocytes in a case of 
chronic hemoptysis. The cytology of the sputum shows little of diagnostic 
value. 

^ Jour. Am. Med. Assn.. 1912, LIX, 1794. 



THE SPUTUM II 

The thin smears of sputum may be treated like blood-smears and stained 
with the same stains when one desires to study the cellular types present. 

(b) Red Blood-cells. 

These cells are frequently found in the sputum and may have much signifi- 
cance. They may occasionally arise from contamination with nasal or buccal 
discharges, but the true bronchial or pulmonary sputum usually shows them 
only in cases of hemorrhage or exudation. The rusty sputum of pneumonia 
contains large numbers of such cells and the hemorrhage from a tubercular 
cavity may be very extensive. These cells are occasionally well-preserved, 
but at times they are difficultly recognizable. They may be very much 
distorted in shape, so that their color and staining properties must be relied 
upon for differentiation. 

(c) Epithelial Cells. 

Various types of epithelial cells are found in the sputum.^ Pavement 
epithehum may come from the mouth, pharynx, and upper larynx. Cylin- 
drical epithelium may be derived from the nose or the bronchi. These cylin- 
drical cells may be ciliated, but rarely does one find these ciliated forms except 
incases of asthma and bronchitis. Alveolar epithelial cells are present in nor- 
mal sputa as they are constantly desquamated from all the epithelial sur- 
faces. These cells assume a large variety of forms and frequently show 
various types of degeneration. They are very numerous in bronchitis and in 
general inflammatory conditions of the lungs, but may occur in almost any 
condition, associated with irritation along the respiratory tract. These cells 
contain large numbers of granules which are probably referable to coal pig- 
ment. Such cells give the sputum a grayish or green color. An abundance 
of such cells in the sputum was designated in earlier times as "phthisis melan- 
otica.'^ Occasionally these alveolar cells are filled with fat globules. In 
other cases one finds the so-called myelin globules, which are irregular in 
shape, often showing concentric lines, with very little refractility, and of a dull 
greenish or blue appearance. The cell may be invisible and only the myelin 
appear as a large irregular mass. Much doubt exists as to the origin of these 
globules, but it is more probable that they represent simply the fatty prod- 
ucts of degenerated protoplasm. These cells are especially frequent in the 
normal morning sputum, in acute or chronic influenza, and in the so-called 
desquamative catarrhal pneumonia, when they appear as small lumps re- 
'Sembling boiled sago. Occasionally free myelin globules are found in the 
sputum. These globules stain poorly with the aniline dyes, are stained 
yellow with iodin, but do not stain black with osmic acid or red with 
Sudan III. 

Frequently these alveolar cells contain pigment granules, derivatives of 
hemoglobin. This material is in the form of amorphous granules of a brown 
color and seems to be identical with hemosidern, but may become iron-free, 
when it more closely resembles hematoidin. Cells containing such granules are 
especially numerous in chronic heart disease and are styled, therefore, "heart- 
disease cells.'' They occur, however, in any condition in which red blood-cells 

^ See Otani, Jour. Exper. Med., 191 7, XXV, 333. 



12 DIAGNOSTIC METHODS 

escape into the alveoli and are found, therefore, in pneumonia, infarction 
of the lung, and hemorrhagic pulmonary tuberculosis. 
(d) Elastic Tissue. 

The presence of elastic tissue in the sputum is indicative of destruction of 
the lung tissue and in many cases is found in the sputum before one can detect 
tubercle bacilli. It has, therefore, some importance in the early diagnosis of 
tuberculosis. When this elastic tissue is grouped in masses it is usually vis- 
ible to the naked eye. However, one more frequently relies upon microscopic 
examination for its detection. The method of Clark, as applied to the de- 
tection of elastic tissue, is usually the best one to follow. The sputum is 
placed upon a glass plate about 14 inches square and pressed out by a smaller 
one, about 6 inches square, into a thin layer. The plates are then placed upon 
a dark background and examined with a hand lens. Instead of these glass 
plates one may use Petri dishes. 

The elastic tissue fibers appear either in the form of distinct strands some- 
times grouped in an elongated network, in that of the alveolar type, in which 
the fibers preserve the outline of the alveoli and are long and branching, or in 
that from the arteries in which we may have a distinct sheet-like arrangement. 
These fibers are characterized by their undulating outline, their curling ends, 
their sharp edges and uniform diameter, their frequent branching, and their 
intense refractility. These characteristics are brought out both with the low 
and with the high power of the microscope. The fibrous tissue fibers are dif- 
ferentiated from elastic fibers by the fact that the former are present in bundles 
of fine wavy lines without the coarse black refractive appearance of elastic 
tissue. Chains of bacteria, especially the leptothrix forms, frequently inter- 
lace in such a way as to simulate the alveolar structure of elastic tissue. These 
chains differ, however, from elastic tissue in their refractility, in the absence 
of the wavy outline, and in their denser arrangement. The elastic tissue 
fibers derived from food substances have the same characteristic as the lung 
elastic fiber, with the exception that they are not arranged in the alveolar form 
and may at times be coarser and more irregular in outline than the pulmonary 
elastic tissue. Vegetable cells and fibers, as well as fatty acid crystals, which 
may occur in the sputum, should not mislead one into assuming the presence 
of elastic tissue. 

Should one wish to stain elastic tissue, he may use the orcein stain of 
Unna-Tanzer. This stain consists of one gram of orcein dissolved in a mixture 
of 80 c.c. of 95 per cent, alcohol and 35 c.c. of distilled water, 40 drops of" 
strong hydrochloric acid being added after solution is complete. In using this 
stain the elastic fibers are treated with a few c.c. of the dye and then warmed 
for five minutes, after which the preparation is decolorized with acid alcohol. 
The elastic tissue fibers will be stained a brownish violet by this process. 

If the elastic tissue is very small in amount we usually resort to chemical 
means for the isolation of this material. Ten c.c. of sputum are mixed with 
an equal volume of 10 per cent, sodium hydrate solution and the mixture is 
boiled until it becomes homogeneous. Four volumes of water are then added, 
the entire mass well mixed, and either allowed to stand or to settle by cen- 
trifugation. In this way the constituents of the sputum are destroyed with 



THE SPUTUM 13 

the exception of the elastic tissue which has, however, become swollen 
and paler and does not have its characteristic appearance. 

About 90 per cent, of cases showing elastic tissue in the sputum are of 
tuberculous origin, according to Dettweiler and Setzer. As the healing proc- 
ess begins and proceeds this elastic tissue gradually diminishes in amount, so 
that a constant presence or an increase in the amount indicates a progressive 
condition. It is seen in abscess of the lungs, in bronchiectasis, in pulmonary 
infarct, and occasionally in pneumonia. It is found in cases of gangrene of the 
lungs, although there are many statements that it is digested by the trypsin- 
like ferment so common in such conditions. Osier states that he has never 
seen a case of gangrene of the lung in which elastic tissue fiber could not be 
found. 
(e) Crystals. 

Crystals are never found in the freshly formed sputum, being indicative 
of stagnation of the material within the body or of decomposition after being 
expectorated. 

(i) Fatty Acid Crystals. 

These crystals occur most frequently in the sputum of gangrene, putrid 
bronchitis and of chronic tuberculosis. They occur as distinct needles, either 
singly or in groups, may be short and relatively thick with pointed ends, or 
they may be long and very closely resemble elastic fiber. Pressure upon the 
cover-glass will produce varicosities in these crystals which do not appear in 
the case of elastic tissue. They are soluble in alkalies and in ether and melt 
into fat globules if the slide be warmed. 

(2) Cholesterin. 

Crystals of cholesterin are found in the sputum of chronic lung abscesses, 
empyema and chronic tuberculosis. They are not as frequent as are the fatty 
acid crystals, but are usually associated with them when they are present. 
These crystals have a distinct rhomboid form with notched angles. 

(3) Hematoidin. 

Crystals of hematoidin occur very rarely in sputum, and then only when 
extravasation has taken place into the alveoli. They are found very rarely 
after direct hemorrhage unless the extravasated blood remains for some time 
in the alveoli. These crystals occur especially in abscess of the lungs, empy- 
ema, or perforating liver abscess. They are rhomboid or needle-shaped 
crystals, ruby-red in color, and may show small curved filaments projecting 
from the angles of the larger forms. 

(4) Leucin and Tyrosin. 

These substances are formed by the decomposition of protein material 
and are found, therefore, in the putrid sputum of an empyema, or from a per- 
forating liver abscess, and in the very early discharges of true lung abscess. 
The tyrosin is found in the form of long refractive needles, frequently ar- 
ranged in bundles, while the leucin appears as distinctly spherical masses with 
concentric striations and radiating lines. 

(5) Magnesium-ammonium Phosphate. 

These crystals appear in the sputum under the same condition as do the 



14 DIAGNOSTIC METHODS 

preceding tyrosin and leucin crystals. They are usually the typical coffin-lid 
crystals so common in decomposed urine, but may assume irregular 
structure. 

(6) Calcium Oxalate. 

These crystals occur in the sputum in conditions associated with decom- 
position and appear either in the typical octahedral crystal with a cross con>- 
necting the corners or as the more atypical dumb-bell shaped crystal. 

(7) Charcot-Leyden Crystals. 

These crystals are apparently derived from the eosinophile cells as they 
are more frequently present only in conditions in which the eosinophiles are 
very numerous, hence the term ''leucocyte crystals." They increase in the 
sputum either from stagnation within the system or after being expectorated. 
They are associated in asthmatic attacks with the spirals of Curschmann, 
being frequently included in the meshes of the spiral! 

These crystals form straight, pointed, colorless, hexagonal, double pyra- 
mids, resembling a very much elongated diamond. They have sharp elon- 
gated points with clear-cut edges, are very brittle, are colorless, show little 
refractility, and vary greatly in size. They may occur singly or in groups, 
forming either clusters or distinct Greek-cross types. They have been 
supposed to be identical with the spermin crystals of Bottcher, but the hex- 
agonal type of the crystal as well as the facts that they do not show marked 
double refraction by polarized light and have but a single optical axis should 
serve to differentiate them from the spermin crystals. They are colored 
yellow with Florence's reagent and may be stained with the polychrome and 
other blood dyes. (See Semen.) 
(/) Bacteria. 
(i) Saprophytes. 

The bacteria found in the sputum are very numerous and under normal 
conditions are purely saprophytic. We may even at times find many truly 
pathogenic organisms in the sputum which are of no clinical significance, 
although one may be led into making a diagnosis without sufficient clinical 
evidence.^ These saprophytes may occur in the fresh sputum or develop 
therein after the specimen has stood for some time. The various chromo- 
genic bacteria are of particular interest as their development along the respira- 
tory tract may so change the color of the sputum that the examiner may be 
led astray. 

Among the ordinary saprophytes found in the sputum we find represen- 
tatives of the strep to thrix^ and the leptothrix groups.^ Flexner and Warthin 
and Olney have reported the presence of a streptothrix, the streptothrix ep- 
pingeri, in the sputum of cases showing the clinical symptoms of pulmonary 
tuberculosis. These organisms are about four times as thick as the tubercle 
bacillus; when stained they are resistant to decolorization by acids, but are 

^Boston (Interstate Med. Journ., 1914, XXI, 330) reports the relatively frequent 
presence of tubercle bacilli in the sputum of acute colds, with disappearance of these organ- 
isms during convalescence. ' 

2 3ee Claypole, Jour. Exper. Med., 191 2, XVII, 99. 

•^ See Kato, Mitt. a. d. med. Fakultat der k. Univ., Tokyo, 1915, XIII, 441. Wherry 
and Olivier, Jour. Infect. Dis., 191 6, XIX, 299. 



THE SPUTUM 15 

slowly decolorized by strong alcohol. Stained specimens are easily made with 
the use of methylene-blue dyes or of Gram's method. The leptothrix group 
is particularly abundant in the mouth and is found in large numbers in the 
lungs in cases of pulmonary gangrene. 




\athc<rir,eM;(( 

Fig. 3. — Aspergillus fumigatus. 

Yeast fungi occur in the sputum at times, but rarely in the fresh speci- 
mens.^ They are oval or elliptical cells and are very refractive, sometimes 
resembling very closely fat droplets. Their appearance may vary from that 
of a simple oval cell without distinct limiting membrane to those with definite 
membrane and vacuoles. These cells are especially characterized by their 




Fig. 4. — Micrococcus catarrhalis. (From Emery's "Clinical Bacteriology.") 



tendency to throw out projections or buds at various points of their periphery. 

They vary in size from i to 40 microns in diameter. While these yeast fungi 

are usually extraneous, cases are reported (Busse) iti which pathogenic yeasts 

have been found in anomalous pulmonary conditions. These organisms stain 

with the ordinary aniline dyes and appear in some instances to be acid-fast 

(resisting decolorization by acid). They are gram-positive. ^ 

^See Breed, Arch. Int. Med., 1912, X, 108; Jour. Am. Med. Assn., 1913, LXI, 472. 
2 See Henrici, Jour. Med. Research, 1914, XXX, 409. Simon, Am. Jour. Med. Sc, 191 7, 
CLIII, 231. 



i6 



DIAGNOSTIC METHODS 



Various types of molds are found in the sputum. Some of these appear 
to be distinctly pathogenic, while the majority are merely saprophytic. 
These molds are found in the true sputum only in cases associated with destruc- 
tive processes of the lungs. There are at present many reports of cases show- 
ing that some of them at least may be distinctly answerable for primary 
infections. 

Among these pathogenic molds we find certain types of the Mucor, of 




Fig. s. 



-Budding forms of blastomycetes found in sputum. 
{From photograph by W. A. Pusey.) 



the 130 varieties of which six are known to be distinctly pathogenic. Besides 
this type we find, as the most important pathogenic mold of the sputum the 
aspergillus fumigatus, 16 cases of pulmonary affection (pneumonomycosis 
aspergillina) having been traced by Sticker to this fungus.^ Other types of 
the aspergilius are the flavus, niger, and the sub-fuscus. The penicillium 
glaucum^ and the Oidium albicans are occasionally found in the sputa. 

Among the bacteria which may be purely saprophytic we may find almost 

^ See von Teckman, Zur Lehre von der Pneumonomykosis aspergillina, Inaug. Dissert., 
Basel, 1913. Ernst, Jour. Med. Research, 1918, XXXIX, 143. 

2 See Weidman, Proc. Path. Soc, Phila., 1915, XVII, 62. Magalhaes, Brazil-Medico 
1916, XXX, 25. 



THE SPUTUM 17 

any of the pus-forming organisms. More frequently, however, when these 
organisms are present in excessive numbers a contamination or a direct patho- 
genic influence should be suspected. A form known as the micrococcus te- 
tragenus occurs both as a pathogenic and a harmless organism. It consists, 
as its name implies, of four cocci arranged in a square within a mucous capsule. 
It stains with the ordinary dyes and is Gram-positive. This organism is 
found, in its pathogenic state, in bronchitis, tubercular cavities, and hemor- 
rhagic infarctions.^ The harmless form differs from the pathogenic type in 
the fact that it cannot be cultivated. 

The sarcinae are rarely found in the sputum. These organisms are 
somewhat smaller than those occurring in the stomach and are probably 
purely saprophytic in the sputum. They are found, however, in cases of 
putrid bronchitis, especially when this occurs in emphysematous lungs, in 
gangrene, tuberculosis, and pneumonia. 

The micrococcus catarrhalis is found frequently in the sputum as a sapro- 
phyte, but may become pathogenic especially in some epidemics of la grippe.^ 
It is larger than the ordinary staphylococcus, while its arrangement in lateral 
pairs and its Gram-negative properties obscure its differentiation from the 
gonococcus and meningococcus unless cultural methods be employed. (See 
last chapter.) 

In cases of general systemic blastomycosis, Eisendrath and Ormsby have 
found the blastomycetes in the sputum. They recommend the examination 
of the unstained specimens after the addition of lo per cent, sodium hydrate 
solution. In such preparations examined with a high-power dry lens, the 
typical refractile blastomycetes are observed. (See Parasitic Diseases.) 

(2) Pathogenic Types. 
(a) Tubercle Bacillus. 

This organism is the most important pathogenic type found in the sputum. 
Its detection is ijeually easy and should be attempted in all suspicious cases, 
as an early diagnosis may frequently save the life of the patient. In the days 
before the organism was recognizable, physicians based their diagnosis of con- 
sumption upon the macroscopic appearance of the sputum. While such 
examinations frequently lead to a presumptive diagnosis of tuberculosis, 
nothing can settle the question except the microscopic examination of the 
sputum. This statement needs some modification in several ways. In the 
first place, specimens of tubercular sputum may not show the presence of the 
bacilli so that several examinations of sputum, collected at different periods, 
must be made. In the second place, the sputum may be examined in the 
very early stage of the disease and no tubercle bacilli be found, but in such 
cases the presence of elastic tissue fiber would be very significant of tubercular 
changes. In these days we have, fortunately, recourse to other diagnostic 
measures in case tubercle bacilli cannot be found in the sputum. I have 
reference here to the use of tuberculin, either introduced in the form of an 

^Steele (Jour. Am. Med, Assn. ,1914, LXII, 930) reports a case of general septicemia 
due to this organism. 

2 Hastings and Niles, Jour. Exper. Med., 1911, XIII, 638. 



l8 DIAGNOSTIC METHODS 

injection as done by Koch, dropped into the eye as advocated by Calmette, 
as advised by Pirquet appUed after the manner of vaccination, or used as an 
inunction as suggested by Moro. 

In examining the sputum microscopically the fine cheesy particles pre- 
viously mentioned are selected and smears of such material made upon glass 
slides. It is always advisable to make at least five such smears to insure 
definite results. It has frequently been found that many suspicious-looking 
sputa show no tubercle baciUi when subjected to the ordinary methods of 
examination. In order, therefore, to increase the possibihty of positive re- 
sults, the sputum should be rendered homogeneous and fluid. The best 
method of accompHshing this is, in the writer's opinion, Loeffler '^^ modification 
of the antiformin process of JJhlenhuth. The technic is as follows: 5, 10, or 
more c.c. of the sputum are placed in a flask and mixed with an equal quan- 
tity of a 50 per cent, solution of antiformin (a 10 per cent, solution of sodium 
hypochlorite containing 5 to 10 per cent, of sodium hydrate) and boiled for a 
period not exceeding 15 minutes. Solution occurs associated with consider- 
able foaming and browning of the mixture. For every 10 c.c. of this solution 
are now added 1.5 c.c. of a mixture of i part of chloroform and 9 parts of 
alcohol. After thoroughly shaking to produce a fine emulsion, portions of 
the fluid are placed in sedimenting tubes, the tubes are corked, and centri- 
f uged for fifteen minutes. The heavier elements collect in a film just above the 
chloroform, which film holds the tubercle bacilli owing to the marked affinity 
of chloroform for the fatty and waxy material in these organisms.^ The 
supernatant liquid is poured off and the film above mentioned is removed and 
placed upon a glass shde, the excess of fluid being taken up with filter-paper. 
As a fixative a drop of egg-albumin, preserved with J^ per cent, carbolic acid, 
is added and a thin spread made by means of a second slide. This smear is 
allowed to dry and is then stained by one of following methods. This enrich- 
ment process of Loeffler furnishes preparations which often show a remarkable 
increase in numbers of tubercle bacilli as compared with those found by the 
usual smear methods. A further advantage of this method is that practically 
all organisms, with the exception of those of the acid-fast type to which the 
tubercle bacillus belongs, are destroyed. If the mixture be allowed to stand 
for a few hours instead of being boiled, one may obtain material for pure-cul- 
ture or for inoculation purposes, as the secondary invaders are ehminated. 
For these latter purposes it is necessary to wash the sediment frequently with 
sterile water by means of centrifugation and decantation to remove the excess 
of alkaH present. This may, of course, diminish the number of tubercle 
bacilli and, at the same time, cause contamination of the specimen. Hence, 
inoculation and cultural experiments frequently give negative results. 

A somewhat different method of eliminating the secondary invaders and of 

1 Deutsch. med. Wchnschr., 1910, XXXVI, 1987. See also, Krauss and Fleming, Jour. 
Lab. & Clin. Med., 1916, I, 919; Giraud et Derrien, C. R. soc. biol., 1916, LXXIX, 976; 
Greenfeld and Anderson, Lancet, 1919, II, 423; Suyenaga, Am. Rev. Tubere., 1919, III, 
473; Goeckel, Med. Record, 1919, XCVI, 804; Greenfield and Anderson, Lancet, 1919, 
II, 423; Woolley, Jour. A. M. A., 1920, LXXIV, 525. 

2 See Panzer, Ztschr. f. physiol. Chem., 1912, LXXVIII, 414; Wells, Interstate Med. 
Jour., 1914, XXI, 221. Burger, Biochem. Ztschr., 1916, LXXVIII, 155. 



THE SPUTUM 19 

obtaining the tubercle bacillus in pure culture from the sputum (as well as 
other material) has been introduced by Petrof. ^ By the employment of a 
special culture medium to which gentian violet is added, Petroff takes 
advantage of the fact that this dye will inhibit the growth of many 
organisms but will not retard the development of the tubercle bacillus at 
the dilution at which it is used. Further, he adopts, as his homogenizing 
agent, an alkaline solution to which the tubercle bacillus is resistant 
while the viability of most other organisms is quite strongly affected. 

Preparation of the Culture Medixim 

Five hundred grams of beef or, preferably, veal are infused in 500 c.c. ot 
a 15 per cent, solution of glycerin in water. Allow the infusion to stand for 
24 hours and squeeze the meat in a sterile press, collecting the juice in a 
sterile beaker. 

Sterilize the shells of one dozen fresh eggs by pouring hot water upon them. 
Carefully break the eggs into a sterile beaker and thoroughly mix the white and 
yolk with a sterile rod. Filter through sterile gauze into a sterile graduate. 

To two parts of the egg add one part of the meat infusion and add to 
this mixture sufficient i per cent. alcohoHc solution of gentian violet to 
make a dilution of i to 10,000. Tube about 5 c.c. of this mixture in each 
sterile test-tube and inspissate the slanted tubes for three successive days: 
on the first day at 85°C. until all the medium is soldified; on the second and 
third days for not more than i hour at 75°C. (If this medium is to be used 
for the isolation of the bovine type of tubercle bacillus, the glycerin may be 
omitted in making the meat infusion.) 

Method 

Equal parts of fresh sputum (about 5 c.c.) and 3 per cent, sodium hydrate 
solution are well shaken and placed in the incubator for 20 to 30 minutes until 
the sputum is fairly well digested. Neutrahze this mixture to sterile fitmus 
paper with normal hydrochloric or sulphuric acid. CentrifugaHze at high 
speed for 10 minutes. Decant the supernatant fluid and spread the sediment 
over several slants of the culture medium. Incubate for 7 to 12 days. 

The cultural characteristics may be more or less variable, some of the 
colonies being of small pin-point size, while others are large and flat. Some of 
the types may decolorize the medium, while others appear violet. Mor- 
phologically, the organisms may vary from cocci to long rods. 

By this method of Petroff tubercle bacilli may be detected in a compara- 
tively short time in cases which have shown repeated negative results by the 
usual microscopic methods, as well as in cases which have not yielded positive 
results with animal inoculation following the antif ormin method. It is simple, 

^Jour. Exper. Med., 1915, XXI 38; Bull. Johns Hopkins Hosp., 1915, XXVI, 275; also 
Mitchell and Simmons, Jour, Am. Med. Assn., i9i5,LXV, 245; Williams and Burdick, Jour. 
Bacteriol., 1916, 1, 411; Keilty, Jour. Exper. Med., 1915, XXII, 612 and Ibid., 1916, XXIV, 
41; Lewis, Ibid., 1917, XXV, 441; Stewart, Ibid., XXVI, 755; Corper, Fiala and Kallen, 
Jour. Infect. Dis., 1918, XXIII, 267; Corper, Am Rev. Tuberc, 1919, III, 461; Wilson, 
Brit. Med. Jour., 1920, I, 146. 



20 DIAGNOSTIC METHODS 

practical and reliable. It is recommended as it tends to increase the 
possibility of diagnosis in obscure cases and may, even, supplant animal 
inoculation tests. 
Staining Characteristics. 

The methods of staining the tubercle bacillus depend upon the property, 
possessed by this organism, of taking up the aniline dyes with great difficulty, 
but, when once stained, of becoming just as resistant against decolorization. 
Breskman (Jour. Am. Med. Assn., 1910, LIV, 1591), Dixon (Ibid., 1913, LX, 
993) and, more recently, Wherry (Jour. Infect. Dis., 1913, XIII, 144) have 
show^n that the tubercle bacillus varies its acid-fast properties in its develop- 
ment on culture media, becoming more acid-fast or non-acid-fast according 
as the cultural conditions are or are not favorable for the synthesis of fat. 
Tamura (Ztschr. f. physiol. Chem., 1913, LXXXVII, 85) demonstrates that 
this acid-fast property is due to the presence in the bacterial cell of an alco- 
hol, mykol (C29H56O), which is partially bound to some higher fatty acid in 
the form of an ester. 
Ziehl-Neelsen Method. 

The smears are made upon glass slides and are fixed by passing several 
times through a flame. The smear is covered with carbol-fuchsin solution^ 
(a mixture of 90 parts of 5 per cent. carboHc acid water and 10 parts of a con- 
centrated alcohohc solution of fuchsin), and is then heated over a flame for 
one to three minutes in such a way that the staining solution steams, but 
does not boil. If the staining solution is heated too strongly the smear decol- 
orizes less readily so that it is very good practice never to boil the staining 
solution. Where several shdes are to be examined, the writer has found the 
copper plate of Ehrlich very useful. ^ The slides are laid upon the plate, are 
covered with the stain, and allowed to heat for 10 minutes. The usual tech- 
nic is, however, to heat a single shde at one time, more of the stain being 
added as the first evaporates. Some workers find that the immersion of the 
smear in cold carbol-fuchsin for 24 hours gives somewhat clearer pictures, but 
the time is too long for the ordinary laboratory diagnosis. Having thus 
stained the smear with the carbol-fuchsin solution, it is then decolorized. 
The tubercle bacillus is not only acid-fast, but also alcohol-fast, so that we 
use decolorizing agents containing both acid and alcohol. There are many 
acid-fast organisms known, such as the bacillus leprae, the smegma bacillus, 
the timothy bacillus, the butter bacillus and many saprophytic bacilU found 
in water, soil, and manure.^ Few or none of these organisms are absolutely 
both alcohol and acid-fast, so that the use of the combined decolorizer will 
usually differentiate the tubercle bacillus. Many decolorizing agents have 
been advised, but the writer finds the use of a 10 per cent, solution of sulphuric 

^ See Verhoeff, Jour. Am. Med. Assn., 191 2, LVIII, 1355. Cerqueira, Brazil-Medico, 1915, 
XXIX, 321; Klein, N. Y. Med. Jour., 1916, CIII, 2i7;Lesieur, Jacquet and Piutenet, C. R., 
soc. biol., Paris, 1919, LXXXII, 251. 

2 See Schmidt, Jour. Am. Med. Assn., 1915, LXIV, 823. 

^Lycopodium spores, cork cells, honey-comb wax and other substances not infrequently 
found in the sputum are acid-fast and may prove very misleading. See Sartory, Bull. I'Acad. 
Med. Paris, 1919, LXXXI, 281, who calls attention to confusing bacteria belonging to the 
oospora group. 



PLATE I. 



.(> 



N 



f 












/K r 



Tubercle Bacilli in Sputum. Ziehl-Neelsen Method. 



THE SPUTUM 21 

acid in 95 per cent, alcohol very reliable. This decolorizing agent does not 
burn the specimen nor does it prevent the morphological characteristics from 
appearing in a clear-cut way. Some workers advise the use of a 2 per cent, 
hydrochloric acid in 80 per cent, alcohol, while others use 25 per cent, nitric 
aci J, followed by alcohol.^ The technic of decolorization is as follows: Wash 
the smear, which has been stained with carbol-f uchsin, in water and flood the 
specimen with the decolorizing solution until only the faintest pink color is 
seen in the smear. It frequently happens that the thicker portions of the 
smear resist this decolorizing so that it may be necessary either to make a 
new smear or to examine merely the portions which have been decolorized. 
After decolorization is complete the specimen is washed in water and counter- 
stained with Loffler's methylene blue (saturated alcohohc solution of methy- 
lene blue 30 c.c, 100 c.c. of a I to 10,000 aqueous potassium hydrate solution) 
for a few seconds, after which the specimen is washed with water, dried be- 
tween filter-paper, and examined with the oil immersion lens. 

In such preparations the tubercle bacilli are seen as bright red rods, some- 
what bent, sometimes much curved and occasionally showing distinct branch- 
ing forms. ^ In a few preparations one may find the curves of the bacillus so 
marked that a very close resemblance to the spirillar forms obtains. These 
organisms occasionally show a distinct beading, giving the appearance of 
bright red cocci. ^ The size of these organisms varies from i}^ to s^i microns 
and about %o microns in width. They may be single or arranged in clumps, 
sometimes in the form of distinct crosses, sometimes parallel, and very fre- 
quently forming acute angles by the joining of two bacilli. 

Gabbet's Method. 

This method is much more simple than the preceding, but is not as reliable. 
By this method the decolorization and counter-staining are carried out in one 
operation. The smears are prepared as previously described and stained with 
the carbol-fuchsin solution. The excess of the staining solution is drained 
off without washing and is replaced by Gabbc't's methylene blue solution 
(methylene blue 2 grams, sulphuric acid 25 c.c, water 75 c.c). This solution 
is allowed to act for one to three minutes and is then washed off with water 
and the specimen dried and examined. The tubercle bacilli will appear as 
bright red rods as previously described, while the other organisms as well as 
the various cellular types will be stained blue. 

This method is not as reliable as is the former, owing to the fact that the 
alcohol-fast bacilli resist decolorization and may confuse one in making the 
diagnosis. Moreover, the use of the strong acid may cause decolorization of 
some of the tubercle bacilli and will, therefore, give rise to wrong ideas. 
These methods are the ones usually followed in routine laboratory work for 
the detection of the tubercle bacillus. 

^ Bourdy (Bull. Sci. Pharmacol., 1918, XXV, 296) advises the use of 10 c.c. strong am- 
monia water in 90 c.c. of 95 per cent, alcohol. Keilty (Jour. A. M. A., 1916, LXVI, 1619) 
uses 30 per cent, hydrochloric acid without alcohol. 

^ See Dixon, Jour. Am. Med. Assn., 1913, LX, 993 and 1294. 

2 See Babes, Berl. klin. Wchnschr., 1914, LI, 501. 



2 2 DIAGNOSTIC METHODS 

Pappenheim's Method. 

The technic of this method is as follows: The preliminary staining is 
carried out with carbol-fuchsin solution as previously outlined. The speci- 
men is then drained and covered with the decolorizing solution, which is made 
by dissolving i gram of rosolic acid in loo c.c. of absolute alcohol, saturating 
the mixture with methylene blue and adding 20 parts of glycerin. This solu- 
tion is drained off slowly and the process repeated several times. The slide 
is washed in water, dried between blotting-paper and examined with the im- 
mersion lens. The tubercle bacilli are stained red, the other organisms blue. 
Much's Method. 

As is well known, certain specimens of undoubted tubercular sputum as 
well as tissues frequently do not show tubercle bacilli when any of the above 
methods of staining are used, owing to the facts that neither is every acid- 
alcohol-fast bacillus the tubercle bacillus nor is every tubercle bacillus abso- 
lutely acid-fast. It is to Spengler ^ and, more especially, to Much ^ that we 
are indebted for a method which will demonstrate these non-acid-fast types of 
tubercle bacilh, which are distinctly granular, frequently appearing as mere 
granules rather than as true bacilli. These granules, under certain unknown 
conditions, change into true bacillary types and vice versa.^ Much's method 
is a modified Gram method, the tubercle bacilh being Gram-positive.^ Pre- 
pare smears as described above. Cover this smear with a carbol-methyl vio- 
let solution (10 c.c. of a saturated alcoholic solution of Gruebler's methyl vio- 
let B. N. mixed with 90 c.c. of 2 per cent, aqueous carbolic acid solution) and 
heat to boihng several times. Wash off stain with water and cover smear 
withLugol's solution (iodine i gram, potassium iodide 2 grams, water 300 c.c.) 
for 5 minutes. Wash with water and treat with 5 per cent, nitric acid for one 
minute and follow this with 3 per cent, hydrochloric acid for 10 seconds. 
Without washing place the slide in a mixture of equal parts of acetone and ab- 
solute alcohol until the smear is colorless. Wash with distilled water and 
counter-stain with i per cent, aqueous solution of safranin for a few seconds. 
Wash in water, dry thoroughly, and examine with the immersion lens. The 
tubercle bacilli and the granular forms appear bluish while the other organ- 
isms are red.^ 
Value of Examinations. 

Brown^ in an able manner has summed up the value of the sputum exami- 
nation for tubercle bacilli. He gives as his reasons for believing that one 
should be guarded in forming an opinion of the prognosis of certain cases the 
following points: (i) Many of the tubercle bacilli may not be stained at all. 

^Deutsch. med. Wchnschr., 1905, XXXI, 1228 and 1353; Ibid., 1907, XXXIII, 337. 

2 Beitr. z. Klin. d. Tuberk., 1907, VIII, 85 and 357. See Meader, Am. Jour. Med. Sc, 
1915, CL, 858; Miller, Jour. Path. & Bacteriol., 1916, XXI, 41. 

^See Korber, Deutsch. med. Wchnschr., 1912, XXXVIII, 1494; Weiner, Munch, med. 
Wchnschr., 1914, LI, 1838; Bruckner, Ztschr. f. klin. Med., 1914, LXXX, 360; Mircoli, 
Gazz. d. osp., 1914, XXXV, 617; Tallo, Gazz. internaz. di med., 1915, XVIII, 241. 

^ See Benians, jour. Path, and Bacteriol., 1912, XVII, 199. 

^See Mas y Magro, Rev. valenc. de cien. med., 1913, XV, 37; Distaso, Lancet, 1920, 
I, 19. 

^ Montreal Med. Jour., 1901, XXX, 769; Jour. Am. Med. Assn., 1903, XL, 514; Effler, 
Ztschr. f. Tuberk., 1916, XXVI, 418. 



THE SPUTUM 23 

(2) Old foci may give off very few and young foci no bacilli at all. (3) By the 
occlusion of a bronchus the contents of a focus may be shut off entirely for a 
time and thus the expelled sputum may contain a large number of tubercle 
baciUi. (4) The organisms may be present one day and not again for months. 
(5) The organisms may be abundant in one part of a specimen and none be 
found in others. (6) Some patients with fatal tuberculosis (caseous pneumo- 
nia or acute miliary tuberculosis) may have no bacilli in the sputum, while in 
other cases the organisms are present even before physical signs obtain. (7) 
In severe cases with bronchitis the secretion of the bronchus will dilute the 
sputum and give the appearance of a reduction in the number of organisms. 
While the number of bacilli in the sputum may thus vary, it is usually in 
direct ratio to the severity of the disease, although for the reasons above men- 
tioned too much rehance should not be placed upon the number of organisms 
found. Brown recommends the use of a somewhat modified Gaffky's table 
in judging of the prognosis in any particular case. The cases are classified as 
follows, being designated by the Roman numerals: 

I. Only one to four bacilli in whole preparation. 
II. Only one on an average in many fields. 

III. Only one on an average in each field. 

IV. Two to three on an average in each field. 
V. Four to six on an average in each field. 

VI. Seven to twelve on an average in each field. 
VII. Thirteen to twenty-five on an average in each field. 
VIII. About fifty on an average in each field. 
IX. About 100 on an average in each field. 
There has been some attempt to base a prognosis upon the form and 
grouping of the tubercle bacilli, the short rods indicating a rapid growth while 
the longer form shows a slower development.^ The continued expectoration 
of large numbers of bacilH would indicate a cavity, while the sudden increase 

I in numbers associated with an increase in the cellular elements would point to 
lung disintegration. A steady decrease over a long period of time would indi- 
cate improvement. It should be stated as a working rule that the finding of a 
single or a very few organisms in the sputum should be looked upon with sus- 
picion, but that an absolute diagnosis should be made only after repeated ex- 
amination has shown the presence of the tubercle bacilli. ^ The worker will find 
in the study of preparations stained as above that artefacts are very common. 
The sputum in tubercular cases rarely shows tubercle bacilH in pure cul- 
ture. One usually finds large numbers of streptococci,^ staphylococci, micro- 

^See Wilson, Lancet, 1914, II, 1 198; Cohn, Beitr. z. Klin. d. Tuberk., i9i4,XXXI, i; 
Kirchenstein, Ibid., 33. 

2 The type of tubercle bacillus found in the sputum is practically always the human 
strain. Only seven cases are reported in the literature of infection with the bovine type 
(see Griffith, Brit. Med. Jour., 1914, i, 1171; Mollers, Deutsch. Med. Wchnschr., 1914, XL, 
1299. Wang, Jour. Path. & BacterioL, 1916, XXI, 14; Eastwood and Griffith (Jour. Hyg., 
1916, XV, 257) report 21 per cent, of bone and joint tuberculosis of the bovine type in a 
total of 261 cases. See, also, Griffith, Lancet, 1917, I, 216; Kendall, Day and Walker, 
Jour. Infect. Dis., 1920, XXVI, 45 and 77. 

^ See Harvey, Jour Med. Res., 1917, XXXV, 279; Corper, Donald and Antz, Jour. 
Inf. Dis., 1919, XXIV, 496; Lucke and Hague, Ibid., 531. 



24 DIAGNOSTIC METHODS 

cocci catarrhalis, and frequently influenza bacilli an.d pneumococci. Pus cells 
may be few or many. Blood-cells may or may not be present, while elastic 
tissue fiber is very frequent, appearing in many cases before tubercle bacilli can 
be demonstrated. The writer has frequently found sputa showing fairly large 
numbers of tubercle bacilli becoming practically negative if the sputum be 
allowed to stand exposed to the air for 48 hours. The explanation is that 
the other organisms so far outgrow the tubercle bacillus that they prevent 
any further development of this latter organism and bring about such degen- 
eration that the staining qualities of the tubercle bacillus are markedly 
affected. This fact has been taken advantage of in the clinical use of in- 
jections of pus organisms into tubercular joints. 

(b) Lepra Bacillus. 

The bacillus leprae, first described by Hansen, is a small slender bacillus 
from 4 to 6 microns in length and surrounded by a slimy envelope. These 
bacilli behave toward staining reagents very much like the tubercle bacillus, 
but are less resistant toward acid and alcohol than is the tubercle bacillus, so 
that a differentiation is possible provided decolorization is rather severe. The 
stained bacilli often show clear spots or appear as if made up of stained 
granules. 

These organisms may be found in many cases of leprosy in the sputum 
or nasal secretion, so that in doubtful cases a differentiation is necessary. 
While these organisms stain much more easily than do the tubercle baciUi and 
are more easily decolorized, it may be necessary to resort to inoculation ex- 
periments to make the absolute differentiation.^ 

(c) Smegma Bacillus. 

This bacillus may be found normally in the saliva, coating of the tongue, 
the tartar of the teeth, and in the crypts of the tonsils. Pathologically, it 
may be found in cases of simple bronchitis, in the sputum in cases of gangrene 
of the lungs, and in the suppurative discharges from the ears. While these 
organisms are much more commonly confused with the tubeTcle bacillus when 
the urine is examined than when the Sputum is investigated, yet they must be 
borne in mind in every sputum examination. It may be necessary to resort to 
inoculation experiments to decide the question, but ordinarily the use of the . 
differentiating stain of Pappenheim (previously described) as well as the fol- 
lowing method of Bunge and Trantenroth may be used. After fixation of the 
smear, the fat is removed by soaking the specimen in absolute alcohol. The 
preparation is now covered with a 5 per cent, solution of chromic acid for 15 
minutes, after whicl^ it is washed with water. The smear is stained with car- 
bol-f uchsin, decolorized with 1 6 per cent, sulphuric acid for three minutes, and 
is then counter-stained for five minutes in a concentrated alcoholic solution of 

^See Duval, Jour. Exper. Med., 1911, XIII, 365; also Duval and Wellman, Jour. Am. 
Med. Assn., 1912, LVIII, 1427; Clegg cultivates this bacillus in symbiosis with amcebas 
and cholera vibrios (Philip. Jour. Sc, Sec. B., 1909, IV, 403); Smith, Lynch and Rivas have 
demonstrated the transmissibility of the lepra bacillus by the bed-bug (Am. Jour. Med. Sc, 
1913, CXLVI, 671). See, also. Wolbach and Honeij, Jour. Med. Research, 1913, XXIX, 
367; Honeij and Parker, Ibid., 1914, XXX, 127. Harris and Lanford, Jour. Med. Res., 
1916, XXXIV, 157- 



THE SPUTUM 25 

methylene blue. This method shows the tubercle bacillus as distinct red, 
while the smegma bacillus is blue. While both of these methods of differ- 
entiation are usually applicable, yet one occasionally finds the smegma bacil- 
lus resisting the action of 16 per cent, sulphuric acid for 30 minutes and 
of strong alcohol for 12 hours. 

{d) The Timothy Bacillus. 

This organism is present in the mouth reaching it through the medium 
of butter and milk, which may contain it in large numbers. These bacilh re- 



/ 



Fig. 6. — Diplococcus pneumoniae. (Williams.) 

sist the decolorizing action of both alcohol and acid to almost the same extent 
as do the tubercle bacilh, but they usually appear as somewhat longer and 
thicker rods. Strangely enough, this organism produces a lesion in guinea- 
pigs which resembles very closely that of true tuberculosis, so that the inocu- 
lation test will not always be conclusive unless other animals are inoculated 
with material froin the first one, in which case no lesions develop in the later 
animals. Fortunately, the cultural peculiarities of this organism are markedly 
different from those of the tubercle bacillus, as the former develops readily on 
the ordinary culture media. 

(e) The Pneumococcus (Diplococcus pneumonise). 

This organism, discovered by Frankel and elaborated by Weichselbaum, 
is generally recognized as the etiologic factor in cases of acute croupous pneu- 
monia, although other organisms not infrequently give rise to this con- 
dition. It is found in large numbers in the sputum and other exudates, 
appearing as a small slighty elongated conical or lance-shaped coccus, 
which shows a marked tendency to occur in pairs (diplococci) with the 
broader ends in apposition. Occasionally it is arranged in short chains 




26 DIAGNOSTIC METHODS 

resembling streptococci.^ In exudates and in the blood this organism is 
usually enveloped by a well-defined hyaline zone or capsule. In clinical 
work it is rarely necessary to stain this capsule to identify the organism 
but if this be desirable it is best done by the method of Rosenow.^ It 
stains well with the ordinary dyes and is Gram-positive. 

Friedlander's Bacillus (bacillus mucosus capsulatus) 
is occasionally found in some cases of lobar and lobular 
pneumonia and occasionally may be considered the 
etiologic factor in such conditions, although it is 
usually a secondary invader.^ These bacilli grow readily 
on artificial media, are encapsulated and stain easily 
with the ordinary dyes but are Gram-negative. 
Fig. 7. — Fried- /^n «^i ▼ « -r^ -.1 

lander's bacillus (/) The Influenza Bacillus. 

(above) : pneumococ- This bacillus, known as Pfeiffer's bacillus, is found 

cus (below) (Greene). . . , ,. , ^ • n • ^1 i 

m the bronchial sputum, especially m the pulmonary 

type of this disease.^ The most characteristic sputum is greenish-yellow 

in color with lumps of pus in nummular form. The organisms are found 

in such sputum as small, short bacilli measuring %o to %o micron in 

breadth by ^{q micron in length. They usually occur singly, but may 

form chains. In the stained specimens these organisms show distinct polar 

stainings, appearing frequently as diplococci. They are stained with dilute 

carbol-fuchsin solution, faintly with the ordinary methylene blue solution, or 

are identified by their Gram-negative characteristics. The best counterstain 

used in the Gram method is either Bismarck brown or safranin, the 

organisms appearing both intra- and extra-cellular. 

ig) The Bacillus Pertussis. 

This organism, discovered by-Bordet and Gengou, and elaborated by 
Klimenko, has been frequently found in the sputum in cases of whooping- 
cough. It resembles very closely the influenza bacillus, appearing as short, 
plump, ovoid bacilli, with rounded ends and lying singly or in small groups 
between the pus and epithehal cells. It stains feebly with the usual dyes 

1 Dochez and Gillespie (Jour. A. M. A., 191 3, LXI, 717) have shown that there exist in 
the sputum of pneumonia or in the fluid obtained by puncture of the lungs in such cases 
four types of pneumococci, which may be separated by immunological reactions. These 
groups have been supposed to differ in their virulence, but, in the recent epidemics, this 
has been somewhat contraverted. See, also, Cole, Arch. Int. Med., 1914, XIV, 56; N. Y. 
Med. Jour., 1915, CI, i and 59; Dochez and Avery, Jour. Exper.Med., i9i5,XXI, ii4;LyaU, 
Ibid., 146; Dochez and Avery, Ibid., XXII, 105; Avery, Ibid., 804; Mathers, Jour. Infect. 
Dis., 1915, XVII, 514; Wollstein and Benson, Am. Jour. Dis. Child., 1916, XII, 254; 
Stillman, Jour. Exper. Med., 1916, XXIV, 651; Hartman and Lacy, Jour. A. M. A., 

1917, LXIX, 2165; Blake, Ibid., 1917, XXVI, 67; Mitchell and Muns, Jour. Med. Res., 

1918, XXXVII, 339; Stillman, Jour. Exper. Med., 1919, XXIX, 251; Loewe, Hirschfeld 
and Wallach, Jour. A. M. A., 1919, LXXIII, 170. 

2 Jour. Infect. Dis., 191 1, IX, i. 

3 See Sisson and Thompson, Am. Jour. Med. Sc, 1915, CL, 713; Sisson and Walker, 
Jour. Exper. Med., 1915, XXII, 747. 

-•See Davis (Jour. Am. Med. Assn., 1915, LXIV, 1814) for a discussion of the role of this 
organism, as well as other members of the hemoglobinophilic group as secondary invaders in 
respiratory infections. Also, Wollstein, Jour. Exper. Med., 1915, XXII, 445. Pritchett 
and Stillman, Jour. Exper. Med., 1919, XXIX, 259; Winchell and Stillman, Ibid., XXX, 
497- 



FLATE II. 



.4 ^^c-- - . 


»v • ♦• .. 


t : 


.•' . •', / / • •••' 


•.• 


• ...-^ K / M/ \ 


■T"'" 


/L -■• \,K i' 


:' ."' 


••••• : . . ) •- -• 


/> -s 


•.,..«• 


£* ..* 


h ': n-' i V • 


r , 




\„ 


:: •• / •• •• 


, 


'■^- L 


' c 



){arharine Hill 



Streptococcus Pyogenes. (Methylene Blue Stain.) 



THE SPUTUM 



27 



often showing bipolar staining and is Gram-negative. This organism is 
rarely intra-cellular and may thus be distinguished from the influenza bacillus. 

{h) Typhoid BaciUus. 

This organism has been found in the sputum in typhoid fever cases show- 
ing a coexistent bronchitis or pneumonia. The sputum is usually hemor- 
rhagic in character and shows the bacilli as short, thick rods, staining with 
the ordinary dyes and negative to Gram's stain. 

{i) Staphylococcus and Streptococcus Pyogenes. 

These organisms are found in practically every sputum examined and can 
be identified only by the use of cultural methods. They stain well with any 
of the aniline dyes and are Gram- 
positive. Their presence in the 
sputum has little pathologic sig- 
nificance.^ 

(j) The Bacillus Pestis. 

This bacillus of bubonic plague 
was discovered by Kitasato and 
Yersin in 1894. It is a short, thick 
bacillus, measuring from 0.8 to 2 
microns in length and from 0.4 to 
0.8 micron in thickness. A capsule 
may be usually made out and the 
stained organism frequently resem- 
bles a diplococcus, owing to the 
intense polar staining with interme- 
diate faint staining. It is Gram- 
negative. 

The bacillus pestis is found in the sputum of persons suffering from the 
pneumonic type of this disease and should be recognized owing to the 
markedly infective character of the material. ^ It may be necessary abso- 
lutely to identify the organism by inoculation and cultural experiments. 

{k) The Bacillus Anthracis. 

The sputum of cases of pulmonary anthrax may contain large numbers 
of these bacilli. These organisms are from 5 to 10 microns in length and 
from I to I )^ in breadth. They are frequently grouped in long segmented 
threads, the segments varying in length, but usually being two or three times 
as long as broad. Occasionally these bacilh may be single, but are usually 
multiple. They form oval spores in the middle of the short segments. The 
organism stains with the ordinary dyes and is also Gram-positive. For 
absolute identification cultural and inoculation experiments, the latter into 

^ The streptococcus viridans is not infrequently found in association^with infections of 
the upper respiratory tract. See Cecil, Arch. Int. Med., 1915, XV, 150. Luetscher 
(Ibid., 1915, XVI, 657) discusses the importance of cultural methods in non-tubercular 
infections. 

2 See Strong and Teague, Philip. Jour. Trop. Med., 191 2, VII , 187. 




Fig. 8. — Bacillus influenza in sputum. 
{Ahhott.) 



DIAGNOSTIC METHODS 



white mice, may be necessary, but the morphological characteristics will 
usually identify it. 

(/) The Bacillus Mallei. 

This organism of glanders is found in the sputum in the pulmonary form 
of this disease. Morphologically, there is nothing characteristic in the ap- 
pearance of this organism beyond the fact of the presence of faintly staining 
areas in the protoplasm of the rather long bacilli. These organisms stain by 
Gram's method as well as with the ordinary aniline dyes. For a final 
diagnosis inoculation into a guinea-pig should be made. 

(w) Actinomyces Hominis (Ray Fungus). 

This fungus, which gives rise to the condition known as lumpy jaw in 
cattle, occasionally infects man, causing pulmonary conditions designated 




Fig. 9. — Actinomyces. (Williams.) 

streptothricosis. The mucopurulent sputum in such conditions contains 
elastic tissue and small sulphur-yellow granules which are visible to the naked 
eye and are the characteristic findings of such cases. Macroscopically these 
granules are yellowish, grayish, or brownish in color, and are sometimes 
abundant and sometimes scarce.^ They are very friable, and when gently 
crushed beneath the cover-glass and examined microscopically appear to 
have broken up into hyaline rounded. masses at the margins of which, on 
close inspection, fine radial striations or filaments or hyaline club-shaped 
bodies, all closely set together, may be seen. The club-shaped bodies are 
variable in size and are composed of a hyaline refringent substance. In 
the granules obtained from the lesions in man the club-shaped formations 
are much less frequently observed than those obtained from the lesions in 
cattle (Mallory and Wright). If cover-glass preparations be made and 
stained with Gram's method, one will usually find isolated and matted 
filaments, many of which may be seen to branch, in addition to longer and 

^ Davis (Jour. Infect. Dis., 1914, XIV, 144) calls attention to actinomyces-like granules 
found in the crypts of tonsils and composed of bacilli, streptococci and spirilla. See, also, 
Rullmann, Miinch, med. Wchnschr., 1914, LXI, 1899; Claypole, Arch. Int. Med., 1914, 
XIV, 104; Davis, Ibid., I; Cope, Brit. Jour. Surg., 1915, III, 55. Pilot and Davis, Jour. 
Iifect. Dis., 1918, XXIII, 562; Waksman, Jour., Bacteriol., 1919, IV, 307. 



THE SPUTUM 



29 



shorter fragments of filaments and fine detritus of the same. If clubs are 
present in the granules they may be found scattered throughout the prepara- 
tions. In this connection see Bridge.^ 
(g) Animal Parasites. 
(a) Amebae. 

Artault has described a unicellular ameboid body which resembles very 
closely a leucocyte when stained, but, while motile, differs in refractility and 
staining quality. This he calls the ameba pulmonalis. In cases of perforat- 
ing liver abscess the true amebae coli may be found in the sputum, and, ac- 
cording to Flexner, in cases of abscess of the jaw communicating with the 

A 




Fig. 10. — Paragonimus westermanii (ventral view); 10 X i. A, oral sucker; B, ceca; 
D, acetabulum; E, genital pore; F, .uterus; G, ovary; H, testicles; /, vitelline glands; 
K, excretory canal; L, excretory pore. {Tyson after Braun.) 

mouth. 2 It should be noted here that these organisms may not be numerous 
so that many slides may have to be examined. 
(b) Flagellates. 
Flagellated organisms, such as the trichomonas pulmonalis and the 
cercomonads, are found in the sputum associated with Dittrich's plugs in 
cases of gangrene, putrid bronchitis, and tubercular cavity formation. The 
trichomonas is probably identical with the trichomonas vaginalis or 
intestinalis.'^ 

^ Jour. Am. Med. Assn., 1911, LVII, 1501; also, Schlegel, Kolle and Wassermann's 
Handb. d. path. Mikroorg., 1912, V, 301; Breed and Conn, Jour. Bact., 1Q19, IV, 585. 

- These are probably identical with the endamebae gingivalis found in the mouth in cases 
of pyorrhea. (See next chapter.) 

' See Ohira and Noguchi, Jour. Exper. INIed., 1917, XXV, 341. 



30 



DIAGNOSTIC METHODS 



(c) Cestodes. 

Not infrequently the lung is the seat of infection with the taenia echino- 
coccus.^ In such cases various foreign bodies, such as fragments of mem- 
branes, scolices, booklets, and cysts, may be found in the sputum. Such 
formations may, also, be found in cases of liver abscess perforating into 
the lung. Any one of the above formations is characteristic of this condition. 
The parasite will be discussed fully in the section on Feces, to which the 
reader is referred. 

The sputum in such cases is usually purulent or mucopurulent and may 
be tinged with blood. This sputum may be expectorated over a long period 
of time and may even contain tubercle bacilli from a coexistent tuberculosis. 
A distinguishing point between pulmonary echinococcus disease and perforat- 
ing liver abscess is that the sputum in the latter is usually bile stained.^ 
{d) Trematodes. 

The most common of this class of animal parasites is the ordinary "lung 
fluke," which has been called also distoma pulmonale, distoma Westermanii 
distoma Ringeri, and Paragonimus Westermanii.^ The eggs of this parasite 
are much more frequently found in the sputum than are the parasites them- 
selves, so that the diagnosis will rest with the finding of these ova. These 
eggs measure from 80 to 100 microns in length and 40 to 60 microns in 
width. They are brownish in color, oval in shape, have a smooth thin 
shell and a lid near one end which is quite characteristic. The parasite is 
from 8 to 10 mm. long, 4 to 6 mm. wide, and is very markedly rounded 
anteriorly, being nearly as thick as broad. 

The sputum in such cases is usually small in 
amount, is very tenacious, and is reddish or rusty due 
to admixture of blood with the mucus. Frequently no 
blood is found, in which case the sputum will still be of 
a yellowish or brown color due to the eggs themselves. 
The sputum, also, contains many spirals, which 
resemble very closely the Curschmann spiral and, 
also, the Charcot-Leyden crystals. ^ 

The eggs of another species of distoma, the dis- 
toma hematobium, has been found in the sputum by 
Manson. Gage^ has reported the finding of the larvae 
of strongyloides intestinalis in the sputum. 




Fig. II.— 
paragonimus 
manii, from 
1000 X I. 
after Braun.) 



>vum of 
wester- 
sputum: 
( Tyson 



V. The Sputa in Disease 

(i) Pulmonary Tuberculosis. 

It has been truly said by Brown that pulmonary tuberculosis has no char- 
acteristic form of sputum. The amount voided may vary from the very 

^See Filia, Pensiero med., 1914, IV, 741; also, Ortali, Gazz. d. osp., 1915, XXXVI, 225. 

2 See Maliwa, Munch, med. Wchnschr., 1914, LXI, 2367. 

3 See Nakagawa, Jour. Exper. Med., 191 7, XXVI, 297; Jour. Parasitol., 1919, VI, 39. 

^ For the mode of infection in these cases see Nakagawa, Jour. Infect. Dis., 1916, XVIII, 
131, and Yoshida, Jour. Parasitol, 1916, II, in. 
^Arch. Int. Med., 191 1, VII, 561. 



THE SPUTUM , 31 

slight type of fibroid tuberculosis to the very abundant sputum of cavity 
formation. It is to be said that the amount of pus will usually depend 
upon the extent of the secondary infection, although caseous degeneration 
may lead to the expectoration of large amounts of material resembling pus. 
In the early cases of pulmonary tuberculosis we may find a small amount 
of sputum which is expectorated only in the morning. This may be very 
tenacious and resemble very much the sago-like sputum previously mentioned. 
Sooner or later depending upon the extension of the disease, there will appear 
small caseous particles which are very suggestive. As ulcerative processes 
proceed, the sputum becomes more profuse, yellowish or greenish in color, and 
muco-purulent in character. In any stage of this ulcerative tuberculous con- 
dition we may find blood in amounts ranging from a few blood-cells to a 
sputum loaded with blood from a hemorrhagic focus. Likewise we will find 
elastic tissue in more or less amount and tubercle bacilli varying from a 
few to many in each field. The color of the tubercular sputum may range 
through all the shades of the spectrum, the greenish shade being associated 
with a most marked decomposition. As stated previously, the most sus- 
picious looking sputa frequently contain no tubercle bacilli. 

(2) Croupous Pneumonia. 

The early sputum of acute lobar pneumonia is usually yellowish-red in 
color and very tenacious in consistency. In some cases the sputum is mucoid 
and abundant for a few days, but soon takes on the characteristic reddish color 
from the presence of unchanged red blood-cells. Its consistency is so great 
that the receptacle may be inverted without allowing any material to run out. 
The characteristic rusty sputum, which is found when the exudation into the 
alveoli is taking place, is homogeneous, glairy, very tenacious, and deep red in 
color. This rusty sputum, while characteristic of pneumonia, is sometimes 
replaced by one ranging in color from a yellow to a green. These colors are 
due to different oxidation products of hemoglobin, and are, perhaps, more fre- 
quently observed in the stage of resolution when the sputum becomes less 
tenacious and more abundant. The greenish sputa in pneumonic conditions 
have some importance. This coloration may be due to a coincident jaundice 
or may arise from delayed resolution, especially when the exudate has been 
particularly hemorrhagic. It is, moreover, sometimes an indication that a 
true tubercular condition has intervened and, hence, that the prognosis must 
be guarded. 

The so-called prune-juice sputum usually indicates a severe type of the 
disease, while at times it may signify merely a beginning resolution.^ 

Fibrinous coagula are found, according to Osier, in every case in which 

search is made. These may vary from very small bronchial casts to very large 

branching types. Curschmann's spirals as well as the Charcot-Leyden 

1 Pacini (Interstate Med. Jour., 1912, XIX, 536) has advanced the following reaction as 
characteristic of early pneumonic sputum: Mix i volume of sputum with 10 volumes of 
distilled water, agitate for 5 minutes and filter. To a test-tube containing 10 c.c. of distilled 
water add 5 drops of a i per cent, aqueous methyl-violet solution. Add to this latter 10 
drops of the sputum filtrate. A positive reaction is shown by the appearance of a distinct 
red color. 



32 DIAGNOSTIC METHODS 

crystals are frequently observed. The characteristic organism of this con- 
dition, the diplococcus lanceolatus of Frankel, is usually found, but has only 
incidental importance, as it is so frequently present in the sputum of normal 
individuals.^ 

(3) Bronchopneumonia. 

The sputum of this disease is rarely characteristic. It partakes of both 
the type of a bronchi tic and a pneumonic sputum. It may, therefore, contain 
much mucus and pus, may be viscid, may be streaked with blood, but is rarely 
so distinctly rusty as in the croupous type of this disease. As the disease is so 
limited in extent it is more or less rare to find an abundant sputum or to 
observe fibrinous coagula. Microscopical examination shows various 
organisms, but nothing diagnostic. 

(4) Acute Bronchitis. 

The sputum in this condition is very scanty in the early stages, is usually 
very tenacious and is expelled with difficulty. This early bronchitic sputum 
is known as ''sputum crudum^^ and consists of practically pure mucin, con- 
taining within its meshes a few leucocytes, red cells, bronchial epithelial 
cells, and a few myelin drops. 

After a few days the sputum is increased in amount, becomes less viscid, 
and assumes the type of a distinct muco-purulent sputum. This sputum, 
called the sputum coctum, contains numerous pus-cells, is yellow or yellowish- 
green in color, shows the presence of large numbers of red cells, as a rule, and 
an increase of the polynuclear leucocytes over the mononuclear form. These 
mononuclear forms are more characteristic of the sputum of true tubercular 
conditions. Fat may be found, either in isolated drops or in large masses. As 
improvement in the condition occurs, the sputum becomes more abundant, 
and more distinctly purulent, and then gradually diminishes until it ceases. 
The sputum of acute bronchitis may give much information as to the course of 
the disease, as the transition from the viscid mucoid sputum through the 
abundant purulent stage to the final cessation is quite characteristic. 

(5) Chronic Bronchitis. 

(a) Simple Chronic Bronchitis. 

In most of these cases the sputum is either very little in amount or is much 
more abundant than in the acute forms. Such cases of simple chronic bron- 
chitis are usually those following the acute type of the disease in which we 
find the expectoration, for long periods of time, of a tenacious, viscid, and 
scanty sputum. Later it may become more abundant and muco-purulent, 
and may have a dark color and a distinctly foul odor. 

In the type of chronic bronchitis associated with cardiac disease we find 
large amounts of blood which may be fresh or changed, giving the typical 
prune-juice appearance. In such sputum we frequently find large numbers 
of the so-called '^heart-disease cell" which have been previously described. 

^See Rosenberger and Dorvvorth, New York Med. Jour., 1913, XCVII, 532. 



THE SPUTUM 33 

(b) Putrid Bronchitis. 

This condition is brought about by dilatation of the bronchial tubes fol- 
lowing a chronic bronchitis. The sputum lies stagnant in these dilated 
bronchi so that it decomposes to a great extent. The sputum in such cases is 
very abundant, is of an ash-gray or brown color, is markedly purulent, and has 
a very disagreeable odor. On standing it separates into the three layers which 
have been previously discussed. In such conditions no elastic tissue fiber is 
found, so that we have here a differentiation from gangrenous or tuberculous 
pulmonary conditions. The sputum in this condition is very similar to that 
found in bronchiectasis, which is usually associated with decomposition of the 
sputum. Whether a diagnosis is possible between a straight putrid bronchitis 
and bronchiectasis is doubtful, if one relies merely upon the sputum. The 
sputum in bronchiectasis occurs usually in the morning and is then very pro- 
fuse. It shows, however, the characteristics of the sputum of putrid bron- 
chitis, but is more commonly associated with the presence of pus, while 50 
per cent, of cases show more or less profuse hemorrhage. 

(c) Fibrinous Bronchitis. 

The chief characteristic of this condition is the expectoration of more or 
less perfect bronchial casts. These may be single or may be distinctly branch- 
ing, showing the arrangement of the entire bronchial tree. This condition 
occurs quite frequently associated with many febrile diseases, but in the dis- 
cussion at this point we have reference to the idiopathic type of the disease. 
The sputum in this latter class of diseases is mucoid and very abundant in the 
earlier stages. After a few days there is expectorated, following a severe 
coughing spell, a bronchial cast. This expectoration is usually tinged with 
blood. Such casts may be expectorated over long periods of time and their 
form may vary as previously described. 

(6) Bronchial Asthma. 

The sputum in bronchial asthma is, perhaps, more characteristic than 
that of any other pulmonary condition. During the paroxysm of asthma 
there may be no sputum, or it may be scanty, consisting of the glairy mucoid 
plugs known as the pearls of Laennec. The sputum contains many eosino- 
phile cells and many alveolar epithelial cells with myelin degeneration. The 
mucoid sputum usually contains large numbers of the spirals of Curschmann 
along with the Charcot-Leyden crystals. In some cases of asthma one finds 
small cylindrical casts of bronchi. Some of these branch while the majority 
are straight and may taper at one end into the central fiber of a true spiral. 
Koessler and Moody (Jour. A. M. A., 191 5, LXIV, 1104) report the presence 
in the sputum of asthmatics of a fusiform anaerobic bacillus together with 
pneumococcus, streptococcus hemolyticus and streptococcus viridans. 
(See also, Walker and Adkinson, Jour. Med. Research, 191 7, xxxv, 391.) 

(7) Influenza. 

The sputum of the pulmonary type of this condition shows in the early 
stages as a very scanty tenacious expectoration. Later it increases in amount. 



34 DIAGNOSTIC METHODS 

becomes muco-purulent and often blood-streaked, and is greenish-yellow in 
color. This sputum contains large numbers of Pfeiffer's bacilli, which have 
been previously discussed. 

(8) Gangrene of the Lung. 

The sputum in this condition is very profuse, is greenish-brown in color, is 
very ofifensive in odor, and is extremely fluid in character. It contains shreds 
of elastic tissue which serve to distinguish it from the sputum of putrid bron- 
chitis or bronchiectasis. This sputum separates, as do other forms of sputum 
which have undergone stagnation and decomposition, into three distinct layers. 

Microscopic examination shows fragments of necrotic tissue varying 
from very minute particles to those several cm. in length. Very few epithelial 
cells or leucocytes are found, but red blood-cells are more or less frequent. 
The bacterial content is usually very high, but nothing characteristic is 
found among these organisms. 

(9) Abscess of the Lung. 

The most characteristic feature of true abscess of the lung or of liver 
abscess which has perforated into the lung is the sudden appearance of a large 
amount of pure pus containing fragments of lung tissue. This material 
usually has the normal odor of pus, but may become offensive, although 
never as markedly so as in gangrene or putrid bronchitis. The sputum of 
the perforating liver abscess is usually distinguished from that of the true 
lung abscess by the so-called "anchovy-sauce" appearance. The color may 
vary, due to the presence of various types of bile pigment. Microscopically, 
bilirubin crystals may be found. 

(10) Perforating Empyema. 

The sputum of such conditions is composed almost entirely of pus and is 
thin and liquid. It contains many hematoidin crystals, but very little elastic 
tissue fiber or other tissue fragments. The odor is usually described as that of 
old cheese in the beginning, but soon becomes offensive owing to decomposition. 

(11) Pneumonoconioses. 

The sputum in these various conditions will depend upon the pigment 
with which the lung has been infiltrated. The expectoration is usually muco- 
purulent, very profuse, and is laden with coal-dust (anthracosis), iron-dust 
(siderosis), with stone-dust, chalk-dust or plaster of Paris (chalicosis), and 
with starch granules (amylosis). 

BIBLIOGRAPHY 

1. Andre. La grippe ou influenza. Paris, 1908. 

2. AuFRECHT. Die Lungenentziindungen. Wien, 1901. 

3. Cornet. Die Tuberkulose. Wien, 1901. 

4. De Jong. Etude histo-chimique et cytologique des crachats. Paris, 1907. 

5. Hoffmann. Die Krankheiten des Bronchien. Wien, 1900. • 

6. Sticker. Lungenblutungen. Wien, 1901. 



PLATE III. 




Leptothrix and Spirocheta Buccalis. (Unstained Specimen.) 



CHAPTER II 

ORAL, NASAL, AURAL, AND CONJUNCTIVAL SECRETIONS 

I. Oral Secretions 
(i) General Considerations. 

The oral secretion is a mixture derived from the various buccal glands, 
the submaxillary, sublingual, parotid, and mucous glands. To this secretion 
has been given the name saliva. It is a colorless, odorless, and tasteless fluid, 
which appears somewhat stringy and frothy, separating on standing into two 
layers, the upper one of which is clear and the lower one cloudy. The function 
of this secretion is to moisten the mouth and throat and, also, to aid in swallow- 
ing the food as well as partially to digest the starchy food through the action 
of a specific ferment (ptyalin) which it contains. The normal daily amount 
of saliva secreted is usually about 1,500 c.c, this quantity varying under the 
influence of many factors, both physiologic and pathologic. The specific 
gravity ranges between 1,002 and 1,009 giving a total solid content of 3 to 12 
grams. Its reaction is alkaline, corresponding to 0.006 to 0.48 per cent, 
of sodium hydrate.^ While the reaction of the saliva is normally always 
alkaline, we occasionally find an acid reaction, especially in children and in 
the early morning hours, due to the production of lactic acid by the bacteria 
which are always present in the mouth. Likewise we find an acid reaction 
especially in conditions associated with acidosis, such states very frequently 
leading to dental caries and to many other irritative conditions of the mouth. 
The recent work of Talbot along this line is especially interesting. 

The chemical composition of the saliva does not have any great clinical 
significance^ with the exception of the presence of the sulphocyanates, the 
nitrites, and the characteristic ferment ptyalin. These substances seem to 
have some importance both from a diagnostic and symptomatic standpoint, so 
that a few remarks may be timely. The presence of potassium sulphocyanate 
(KCNS) is more or less characteristic of normal saliva and may be detected 
as follows: Collect a few c.c. of saliva before meals and allow this to filter. 
Add a few drops of hydrochloric acid and then a drop or two of ferric chlorid 
solution, when a distinct red color will be observed, whose depth will depend 
upon the amount of sulphocyanate present. It has been stated that heat 
should be appHed in this test, but the writer has never found it necessary as 
the characteristic reaction almost invariably appears in the cold. This color 
disappears on the addition of mercuric chlorid solution, which fact may serve 
to differentiate it from the similar one given by the saliva of opium habitues 
and due to meconic acid. Very Httle pathologic significance has been at- 
tached to variations in the amount of the sulphocyanate in the saliva, but 

/ Michaelis and Pechstein (Biochem. Ztschr. 1914, LIX, 77) have found the Ph value of 
sahva to be 6.9, that is, almost neutral from the physico-chemical standpoint. 

2 See Herzfeld and Stocker (Zentralbl. f. inn. Med., 1913, XXXIV, 753) for a discussion 
of uric acid in saliva. 

35 



36 DIAGNOSTIC METHODS 

it is interesting to note that in many cases of diabetes as well as in cases of 
severe stomatitis this substance is frequently absent. 

The nitrites may be detected by the more delicate tests used in water 
analysis, as their amount is usually not sufficient for the ordinary quahta- 
tive tests. A very good test is the use of the Griess-Ilosvay reagent (3^ gram 
of sulphaniHc acid is dissolved in 150 c.c. of dilute acetic acid and treated 
with ^{0 gram of naphthylamin dissolved in 20 c.c. of boiling water. On 
standing, a blue sediment forms which is separated and dissolved in 150 c.c. 
of dilute acetic acid). On treating 10 c.c. of saliva with a few drops of this 
reagent and heating, a red color will develop in the presence of nitrites. 

The most important constituent of saliva is the ptyalin which has a defi- 
nite hydrolytic action upon starch, converting this polysaccharid into maltose 
through the intermediate stages of erythrodextrin and achroodextrin. This 
action may be readily seen by treating a httle starch paste with a few c.c. of 
filtered saliva and placing the vessel in the incubator for 10 to 15 minutes. 
At the end of this time iodin solution is added when a distinct red color or an 
entire absence of color will be noticed. It is to be remembered here that starch, 
treated with iodin, is colored blue. Careful work by Litmanowicz^ has show 
that the diaslatic power of saliva is unaffected by physiologic or pathologic 
variations in general body functions. ^ 

(2) Microscopic Examination. 

On allowing saliva to stand it separates into two distinct layers, the upper 
one clear and containing the liquid portion, w^hile the lower is cloudy and con- 
tains the morphological elements. In the microscopic examination of this 
lower layer we observe many epithelial cells in the form of large, irregular, 
squamous cells which are derived from the mucous membrane of the mouth 
and tongue. The number of these cells present depends, of course, upon the 
erosion to which the mouth has been subjected by various irritants either of 
the food or of disease. The characteristic cells of the saliva are the sahvary 
corpuscles, which resemble the leucocytes, but are larger and more granular. 
Occasionally red blood-cells are seen, but these have no direct significance 
other than to denote ulcerative or markedly irritative conditions somewhere 
in the nasopharynx. Beside these constituents of the saliva, we find, micro- 
scopically, many micro-organisms of the mold, yeast, and bacterial types. 
The bacteria are always present in the mouth as they are taken in with the 
air, food, and drink. Few of these have any direct significance, although the 
spirochaeta buccahs and microdentium should be borne in mind, especially 
when an examination is being made for the spirochaeta paUida.^ The former 
is differentiated from the latter by the fact that its ends lie upon a line drawn 
longitudinally through the center of its spirals, while such a fine drawn 
through the pallida Kes above and below its ends. Moreover, it should be re- 

^ Zentralbl. f. d. ges. Physiol, u. Path, des Stoffw., 1909, IV, 81. 

2 See Hirata, Biochem. Ztschr., 191 2, XL VII, 167; vanHaeff, Nederl.Tijdschr. v. Geneesk., 

1915, LIX, 307; Lubimoff, Russk. Vrach., 1915, XIV, 269. Biedermann, Fermentforsch., 

1916, I, 385; Rockwood, Jour. Am. Chem. Soc, 1919, XLI, 228; McGuigan, Jour. Biol. 
Chem., 1919, XXXIX, 273; Grimbert, Jour. Pharm. Chem,, 1919, XIX, 244. 

2 See Thibaudeau, Jour. Am. Med. Assn., 191 2, LIX, 446. 



( 



ORAL, NASAL, AURAL, AND CONJUNCTIV^AL SECRETIONS 37 

membered that the smegma bacillus is an occasional habitant of the mouth 
and throat and may occur in specimens of sputum, giving rise to the assump- 
tion of the presence of tubercle bacilli unless proper means of identification are 
used. Simon has pointed out an interesting fact that the majority of the 
micro-organisms which are constantly present in the mouth cannot be culti- 
vated on artificial media, while the temporary invaders easily develop. 
Many pathogenic bacteria have been found in the mouth of the healthy sub- 
ject.^ This is interesting clinically as showing the constant danger to which 
we are all subject, in case our resistance becomes lowered. The writer recalls 
that the most"virulent culture of pneumococci obtained from 200 throats, both 
diseased and normal, was from his own at the time when he was in perfect 
condition and showed no symptoms thereafter. Beside the pneumonia organ- 
ism, streptococci and diphtheria bacilh are frequently found in the mouths 
of perfectly healthy individuals. Molds and yeast fungi are rarely found in 
the saliva during health, but they are present in pathological conditions. 
(3) Pathologic Changes. 

The normal daily secretion of the saliva is, as stated above, about 1,500 
c.c. The composition of the secretions of the various glands, which contribute 
to the mixed secretion, differs rather widely, the one from the other. We 
may, therefore, have changes, not only in amount of saliva, but, also, in the 
quality, depending on the diseased condition of one or more of these glands. 
The quantity of saliva is diminished in inflammation of the salivary glands, 
such as in parotitis, in all febrile diseases, in diabetes, and in nephritis. The 
secretion is also diminished by the therapeutic use of preparations of bella- 
donna and of opium. It is increased by certain poisons, such as pilocarpin 
and mercury, by excessive irritation with acids and alkalies, and, also, by irri- 
tations arising from carious teeth. Occasional cases have been reported of a 
greatly increased amount of saliva through some obscure nervous reflex, while 
such a condition is not unusual in pregnancy. An increased flow of saliva is 
known as salivation or ptyalism. In determining whether or not salivation 
really exists, observation will frequently show increased amounts of saliva at 
all times. In some cases, hoWever, it is necessary to measure the amount and, 
also, to make later chemical examinations of the saliva. The best way of ob- 
taining saliva free from contamination is to wash the mouth thoroughly 
with a solution of sodium bicarbonate, brush the teeth thoroughly with the 
same solution, and then rinse out the mouth with cold water. On now touch- 
ing the inner surface of the teeth or the edge of the tongue with a glass rod that 
has been dipped into dilute acid, saliva will be seen pouring into the mouth 
from many points. This saliva is then collected in clean receptacles and the 
quantity measured. 

Variations in the reaction of the saliva are not uncommon in pathologic 
conditions. In various intestinal diseases with which we may have an associ- 
ated stomatitis, an acid reaction is frequently noted. Also in fevers, diabetes, 
starvation, and other conditions giving rise to acidosis (overloading of the sys- 

^ See Kiister, KoUe and Wassermann's Handbuch, 1913, VI, 435; Moorehead, Jour. 
A. M. A., igi6, LXVII, 845; Billings, Ibid., 847; Irons, Ibid., 851. 



T,S DIAGNOSTIC METHODS 

tern with acid products) the reaction of the sahva is always acid. Strauss 
and Cohn beheve that the sahva is alkahne, even under pathological 
conditions. 

Coating of the Tongue. 

A coating of the tongue is practically always abnormal, as the normal 
appearance is a bright reddish color with no visible deposits.^ A change in 
the normal appearance of the tongue has so long been indicative, in the minds 
of the profession, of disturbed conditions not only in the mouth, but in the 
stomach and the bowels, that one should always take into consideration any 
such change. In severe infectious fevers a brownish coating with a furred ap- 
pearance is practically always seen. This consists of remnants of food and of 
incrusted blood, along with large numbers of micro-organisms^ and dark des- 
quamated epithelial cells. The white coating contains no blood and is more 
indicative of simple gastro-intestinal disturbance than is the brown coating. 
The so-called "tartar" which forms upon the teeth seems to consist of depos- 
ited calcium carbonate and contains many actively motile spirochete as well 
as large segmented leptothrices, along with leucocytes and epithelial cells. 

Pharyngomycosis Leptothrica. 

In many pathological conditions of the throat, such as tonsillitis, diphthe- 
ria, and thrush, we frequently find the tonsillar and other buccal structures 
covered with a coating which is, in many cases, a distinct membrane contain- 
ing the pathogenic organisms in large numbers. Many perfectly normal sub- 
jects complain of the formation, in the tonsillar crypts, of plugs of material 
which are easily removed by pressure. These are frequently found in patients 
subject to tonsilHtis, but, also in those showing no pathological conditions of 
the tonsils and are closely related to Dittrich's plugs, which have been 
discussed. 

In the pyoid masses of pharyngomycosis leptothrica, one finds large num- 
bers of lymphocytes, epithelial cells and long segmented fungi, the lepto- 
thrices buccahs, which are colored bluish-red by a solution of iodopotassic 
iodid. In such conditions the poly nuclear neutrophiles are present in only 
small numbers. In some cases patches of these fungi extend over quite an 
area of the tonsils so that the appearance may be one of the formation of a 
diphtheritic membrane, although microscopic examination will at once clear 
up the diagnosis.^ 

Diphtheria. 

One of the most important examinations of the oral cavities consists in 
the detection of the diphtheria bacillus (Klebs-Lofiier bacillus), as an early 
diagnosis of this disease frequently enables the physician to institute anti- 

^See Schilling, Deutsch. Arch. f. klin. Med., 1914, CXIII, 622. 

2 Catacuzene (C. R. soc. biol., Paris, 1914, LXXVII, 449 and 452) describes a pleomor- 
phic organism in the scrapings of the tongue in scarlet fever. In smears stained by Giemsa 
stain several forms are seen, minute ovoid or elongated bodies stained an intense blue and 
presenting at the center or near one pole a chromatic spot. 

'Conlon (Jour. Am. Med. Assn., i9i3,LX, 900) has reported the aspergillus niger as a 
causative factor in infection of the pharynx. Basile (Policlinico, 191 7, XXIV, 88 and 97) 
reports a study of pharyngeal blastomycosis. 



PLATE IV. 



^ 






Yathdrine-Hiff. 



Diphtheria Bacilli Showing Polar Staining, (Neisser Method, Counter Stained 

WITH Safranin.) 



ORAL, NASAL, AURAL, AND CONJUNCTIVAL SECRETIONS 39 

toxin treatment. Such an examination should never be omitted in any case 
of suspected sore throat, especially where any membraneous patches are pre- 
sent. ^ By means of a stout platinum loop or a swab of cotton a piece of mem- 
brane or a portion of the exudate is scraped from the throat. This material 
is then spread over the surface of Loffler's blood serum and is allowed to incu- 
bate at 37°C. for six to eight hours. This period of incubation is of some im- 
portance as it has been definitely shown that at the end of six to eight hours 
the diphtheria organism is the only one which will attract much attention, 
while if left for a longer time, other organisms, especially the streptococcus 
and staphylococcus, will so far outgrow the diphtheria bacillus that this latter 
may be unrecognizable unless the incubation be carried 36 hours, when the 
diphtheria bacillus then assumes the ascendency. From this culture, cover- 
glass preparations are then made and stained for one to five minutes in 
Loffler's alkaline methylene blue solution. They are then rinsed in water 
dried, and examined with the immersion lens. 

Neisser's Stain. 

This stain is supposed to differentiate the diphtheria bacillus from all 
others. The smear is stained for five minutes with a methylene blue solution 
(methylene blue, Griibler, i gram, 20 c.c. of 96 per cent, alcohol, glacial acetic 
acid 50 c.c, and water 950 c.c). The stain should be filtered before use. 
The specimen is heated gently during the staining process and the dye re- 
newed as the stain evaporates. Wash in water^ and stain for two minutes 
with an aqueous solution of Bismarck brown or, better, a dilute solution of 
safranin. The polar bodies will be stained a deep blue, while the body of the 
bacillus will take a light brown or red color. ^ 

Microscopically, the stained organism appears as a slightly curved rod, 
but especially characteristic are ^he bizarre forms, such as rods with alternate 
staining and nonstaining portions, rods with distinct deeply staining polar 
bodies, club-shaped or '' narrow- waisted'^ rods, many of which lie together in 
distinctly parallel lines. 

Diphtheria bacilli may be found in the throat for weeks after all symptoms 
have disappeared"^ so that it is wise to enforce isolation of the patient until a 
negative examination for these organisms is obtained." In causing the dis- 
appearance of these bacilli from the throats of " carriers" the method of spray- 
ing the nose and throat every three hours with a 24-hour bouillon culture of 
staphylococcus aureus, as advanced by Schiotz (Ugesk. f. Laeger, 1909, LXXVI, 
1373), has proven very satisfactory. However, Bissell (Jour. Am. Med. 
Assn., 1913, LXI, 1393) believes the method is questionable as it leads to false 
security owing to the fact that the few diphtheria bacilU actually left in the 

^ See Neisser and Gins, KoUe and Wassermann's Hand. d. path. Mikroorg., 191 2, V, 931. 

2 Gins (Deutsch. med. Wchnschr., 1913, XXXIX, 502) advises a short (2 to 3 seconds) 
treatment at this point with Lugol's solution containing i per cent, of lactic acid. See, also , 
Tamura, Ztschr. f. physiol. Chem., 1914, LXXIX, 289; Neilsen, Hospitals tid., i9i5,LVIII, 
225. 

^See Raskin, Deutsch. med. Wchnschr., 191 1, XXXVII, 2384; also, Ponder, Lancet, 
1912, CLXXXIII, 22; Kinyoun, Am. Jour. Pub. Health, 1915, V, 246; Mood, Southern 
Med. Jour., 1915, VIII, 482. 

* See Markl and Pollak, Wien. klin. Wchnschr 1913, XXVI, 1617. 



40 



DIAGNOSTIC METHODS 



deeper structures are masked by the numerous organisms of the sprayed 
culture.^ 

Occasionally in examination of smears from the throat, true diphtheria 
bacilli may be confounded with pseudo-diphtheria bacilli, -and in examination 
of other specimens, such as those taken from the eye, the bacillus xerosis may 
be confusing. These different organisms are best differentiated by the study 
of their action in fermenting or not fermenting certain sugars. According to 
Knapp, the pseudo-bacilli will ferment none of the sugars, the diphtheria bacilli 
will ferment dextrose, mannite, maltose, and dextrin, but not saccharose, 
while the xerosis bacillus ferments dextrose, mannite, maltose and saccharose 
(cane sugar), while it does not ferment dextrin. (See Chapter XI.) 

Vincent's Angina (Ulceromembranous Angina and Stomatitis). 

In this condition, smears taken from the throat, as well as the free sahva 
will be found to contain many organisms of two special types, the first, spirilla, 







Fig. 12. — Vincent's spirillum and bacillus. (Coplin.) 

and the second, long fusiform bacilU. Usually both of these types are found 
together, but occasionally the spirilla are absent. The spirilla usually meas- 
sure from 36 to 40 microns in length and }'2 micron in breadth, while the ba- 
cilli are 6 to 1 2 microns in length and are somewhat thicker in the center than 
at the end. These organisms may be stained with Lofiler's methylene blue, 
gentian violet, or dilute carbol-fuchsin, but they decolorize with Gram's 
method.'^ 

^See Womer, Jour. Am. Med. Assn., i9i3,LXI, 2293. Hektoen and Rappaport (Jour. 
Am. Med. Assn., 1915, LXIV, 1985) advise the use of kaolin, either swallowed or given as 
an insufflation, for this purpose. See, also, Perkins, Miller and Ruh. Jour. Infect. Dis., 1916, 
XVIIT, O07; Kolmer and Moshage, Ibid., XIX, I; Geiger, Kelly and Bathgate, Jour. A. M. 
A., 1916, LXVI, 645; Ott and Roy, Ibid., 800; Friedberg, Ibid., 810; Goff, Ibid., 941; Ruh, 
Miller and Perkins, Ibid., 941; Rappaport ,Ibid., 943; Lewis, Ibid., 1535; Rabinoff, Ibid., 
LXVII, 1722; Kolmer, Woody and Moshage, Am. Jour. Dis. Child., 1916, XI, 257; Walthall, 
Ibid., XIL 149; Weaver, Jour. Infect. Dis., 1917, XX, 125. 

2 See Meader, Jour. Infect. Dis., 1919, XXIV, 145. 

^The organism has been recently cultivated, but inoculation experiments have been 
unsuccessful. See Gins, KoUe and Wassermann's Handb. d. path. Mikroorg., 191 2, V, 1003 . 
See McClintock, Am. Jour. Med. Sc, 191 7, CLIII, 256; Campbell and Dyas, Jour., A. M. A., 
191 7, LXVIII, 1596. This organism is more properly speaking, to be classed among the 
spironemata. 



ORAL, NASAL, AURAL, AND CONJUNCTIVAL SECRETIONS 



41 



Streptococcic Sore Throat. 

During recent years several milk-borne epidemics of sore throat with se- 
vere constitutional symptoms have occurred, especially in Boston, Chicago 
and Baltimore, of which the causative organism is a peculiar streptococcus 
(the streptococcus epidemicus). In smears from the throat and tonsillar exu- 
dates this highly virulent organism occurs in short chains, the spherical cocci 
appearing in twos in the chain. They are strongly Gram-positive and are sur- 
rounded by a definite capsule. They produce a relatively narrow zone of 
hemolysis on blood agar but little or no greenish color. ^ 

Gonorrheal Stomatitis. 

In this condition the usual changes of infection are observed along with 
the appearance of the gonococci in the smears. Boston reports several cases 
of supposed gonorrheal stomatitis in which cultural methods showed the 




Fig. 13. — Oidium albicans. {Kolle and Wassermann.) 

absence of this organism, although the smears showed the presence of intra- 
cellular Gram-negative diplococci. Such reports are not surprising in view 
of the fact that so many saprophytic diplococci (such as the diplococcus fiavus 
and crassus) are found which may or may not stain by Gram's method. The 
almost constant presence of the micrococcus catarrhalis must be borne in 
mind. 
Thrush. 

This is a condition most commonly seen in children, but may occur in 
adults, especially in those with tubercular tendencies. The saliva in this con- 

^ See Davis and Rosenow, Jour Am. Med. Assn., 191 2, LVIII, 773; Hamburger, Ibid., p. 
nog; Davis, Ibid., pp. 1283 and 1852; Capps and Miller, Ibid., p. 1848; Heinemann, Ibid.,^ 
1912, LIX, 716; Leutscher, Ibid., p. 869; Frost, Pub. Health Reports, 1912, XXVII, 1889; 
Stokes and Hachtel, Ibid., p. 1923; Winslow, Jour. Infect. Dis., 191 2, X, 73; Hamburger, 
Bull. Johns Hopkins Hosp., 1913, XXIV, i; Capps, Jour. Am. Med. Assn., i9i3,LXI, 723; 
Mann, Jour. Infect. Dis., 1913, XII, 481; North, White and Avery, Ibid., 1914, XIV, 124; 
Capps and Davis, Jour. Infect. Dis., 1914, XV, 130; Arch. Int. Med., 1914, XIV, 650" 
Rosenow and Moon, Jour. Infect. Dis., 1915, XVII, 69; Kirkland (Brit. Med. Jour., 1914, 
I, 419) reports a case from Cheltenham which belongs in this group; Bray, Jour. Am. Med. 
Assn., i9i5,LXIV, 1127; Krumwiede and Valentine, Jour. Med. Research, 1915, XXXIII, 
231; Winslow and Hubbard, Jour. Infect. Dis., 1916, XVIII, 106; Rosenow and Hess, Jour. 
A. M. A., 1917, LXVIII, 1305 ;Henika and Thompson, Ibid., 1307; Davis, Jour. Infect. Dis.,. 
1918, XXIII, 559; Howard and Orcutt, Jour., Exper. Med., 1920, XXXI, 49. 



42 DIAGNOSTIC METHODS 

dition is usually acid and somewhat increased in amount. Microscopic ex- 
amination of the membrane shows many epithelial cells, leucocytes, and much 
granular detritus with a network of branching band-like formations, showing 
distinct segments.^ The contents of the segments are clear and usually con- 
tain two highly refractive granules, one at each pole. This organism is known 
as the Oidium albicans. It stains well with the ordinary aqueous methylene 
blue solution. 

Oral Endamebiasis. 

The condition, known as pyorrhea alveolaris or Riggs' disease, has for 
years been regarded as a form of bacterial infection in which local irritative 
processes and systemic disturbances are looked upon as predisposing factors. 
Many types of bacteria, such as streptococci, pneumococci and staphylococci, 
have been found and isolated from the pus pockets about the roots of the 
teeth, but these cannot be regarded as the only etiologic factors in this condi- 
tion, at least in the majority of cases. ^ 

While it has been known for years that parasites, especially those of the 
ameba group, were very frequently found in the mouth and in the soft tartar 
about the teeth, these were regarded as innocuous invaders, except in a few 
isolated cases. During the last year a great amount of work, following the 
first communication of Barrett, has centered about these amebae and their 
association with the lesions of pyorrhea, until at present their pathogenic im- 
portance is being freely discussed. There is little question, according to 
Smith and Barrett, that we must now ''regard these parasites as either di- 
rectly causative of a large class of gingival and alveolar pyorrheas or as impor- 
tant members of a symbiotic chain with one or other of the numerous asso- 
ciated vegetable micro-organisms, in the production of these lesions." It is 
to be said that the proof of the pathogenic importance of the endamebae in Riggs' 
disease rests upon their almost constant presence in the suppurating pockets 
of pyorrhea (while they are rarely found in healthy mouths), and the prompt 
removal of both suppuration and of the endamebae when emetin (first advo- 
cated by Roger) is administered locally or generally. The organisms have not 
yet been cultivated, so that the question of conformity with the classic postu- 
lates of Koch has not been investigated. Further, many of the cases supposedly 
cured by emetin show recurrences after a few weeks' freedom from treatment. 
While there is no question of the presence of these amebae in cases of pyorrhea, 
it has not been firmly estabhshed, as yet, that they are truly pathogenic rather 
than merely "members of the symbiotic chain." 

Several forms of endamebae have been described as habitants of the buccal 
and dental tissues, some of which were supposed to be pathogenic. These 

^ Ashford (Jour. Am. Med. Assn., 1915, LXIV, 810 and 1893) reports the finding, in 
cases of sprue, of a yeast (monilia psilosis) not hitherto described. See Ashford, Am. Jour. 
Med. Sc, 1915, CL, 680; 1916, CLI, 520 and 1917, CLIV, 157; Michel, Ibid., 177, and 
Wood, Ibid., 1915, CL, 692 and Jour A. M. A,, 1919LXXIII, 165. 

2 See Nodine, Dental Cosmos, 1914, LVI, 969; Gilmer and Moody, Jour. Am. Med. 
Assn., 1914, LXIII, 2023; Billings, Ibid., 2024; Mayo, Ibid., 2025; Rosenow, Ibid., 2026; 
Craig, Ibid., 2027; Hartzell and Henrici, Ibid., 1915, LXIV, 1055; Hoxie, Jour. A. M. A., 
1915, LXV, 1908; Lescohier, Ibid., 1917, LXVIII, 414; Medalia, Ibid., 798. 



PLATE V 









V. 






















'^*J 


i 


0. 


t 
f 






1 




c 















Camera Llcida Drawings of Endameba Gingivalis (Gros), Stained with Iron Hematoxylin. 
{A. J. Smith and M. T. Barrett, Jnl. of Parasitology.) 



ORAL, NASAL, AURAL, AND CONJUNCTIVAL SECRETIONS 43 

various amebae are more or less similar in their general characteristics, so that 
it is more than probable that the one, now first recognized by Barrett and a 
little later by Bass and Johns as almost constantly associated with pyorrhea, 
is the same as was long ago described by Gros, Steinberg, Grassi, Flexner and 
Prowazek. It is no more than just, therefore, that this ameba, whose patho- 
genicity is being investigated, should be styled Endameha^ gingivalis (Gros) 
and not Entameba huccalis (Prowazek), as some call it. 

These endamebas, as studied by Smith and Barrett, show the following 
characteristics: "Naked parasitic amebse of usual diameter in resting exam- 
ples of 0.030 to 0.035 "^i^- (^with exceptional instances reaching 0.040 or 
slightly above) ; with refractile and faintly greenish-tinted hyaline ectosarc 
well defined from the granular endosarc, but sometimes so thin as to be easily 
overlooked; endosarc granular, colorless and in all but the more minute exam- 
ples containing few of many digestion vacuoles in which globular detritus of 
leucocytic nuclei and red blood cells are commonly found along with bacteria; 
with a small (0.002 to 0.005 rnni-) rounded nucleus, invisible or at best un- 
certainly distinguishable in the unstained specimen, usually central or sub- 
central in position, but at times eccentric (when the ingesta push it to one 
side). The nucleus is very poor in chromatic substance, vesicular, with a 
small "binnenkorper," sometimes showing a minute centriole, a clear space be- 
tween it and the nuclear border containing no chromatin or at most a very 
few incomplete threads; and the border is represented by a thin but somewhat 
irregular line of chromatin, about which the writers are unable to recognize a 
further membrane. The degree of motility manifested by the parasite is 
fairly comparable to that of the dysenteric endameba. The pseudopods as a 
rule are few (one, two or three), usually broadly lobose and commonly attain- 
ing a maximum length of the diameter of the endosarc. The pseudopods are 
composed practically entirely of the ectosarc, the granular endosarc terminat- 
ing at the base or extending but a little into the larger ones." 

The material for examination is best obtained by scraping the bottom 
of the lesion in the peridental membrane between the tooth and alveolar 
process, as the endamebae do not live on the normal gum margin but at the 
edge of the tissue which they are invading. As the secondary infection with 
pyogenic organisms causes a profuse flow of pus which distends the pocket, 
very few endamebae can reproduce and live in this necrotic material. For 
this reason it is absolutely essential for success in this examination that most 
of this purulent material be removed from the old lesions and the specimen for 
examination be obtained from the bottom of the lesions. By means of a tooth- 
pick or, preferably, a Younger scaler No. 8 or 9, remove some of the material 
from the bottom of the lesions. If the specimen is to be examined in the fresh 
condition, mix the material with a drop or two of slightly warm normal saline 
solution on a warm slide and place over this a cover-glass. Examine at once 
with the high-power dry lens. Careful study will reveal the motile organisms 
showing the characteristics mentioned above. If the organisms are not 

1 The name endameba was first suggested by Leidy in 1879 and should take precedence of 
the term entameba advocated by Casigrandi and Barbagallo in 1897. 



44 DIAGNOSTIC METHODS 

actively motile, it is not the simplest matter to recognize them in the fresh 
state. 

For general work the stained specimen is, probably, more satisfactory for 
examination, as differentiation is more certain. Obtain the material as above 
mentioned and scrape it onto a clean slide with a toothpick, spreading it out 
into a thin layer with the flat side of the toothpick. Care should be 
taken not to rub the material back and forth as this may break up the amebae. 
Allow the sHde to dry in the air and fix by passing once or twice through the 
flame. Cover the smear with carbol-fuchsin for loto 15 seconds, wash off the 
excess of stain with water and stain withLoffler's methylene blue for 30 seconds. '■ 
Wash in water, dry and examine with the immersion lens. Properly pre- 
pared specimens should have a deep purple color. The red blood cells stain 
a deep red. Pus cells show a bright purple nucleus with a Hght pink proto- 
plasm. Tissue cells are larger than the pus cells and appear reddish with a 
small purple nucleus. Bacteria may be stained either red or blue, depend- 
ing on their type. The endamebse vary in size from cells somewhat smaller 
than a pus cell to those several times as large. The endoplasm stains a deep 
blue, while the irregular border of ectoplasm stains purple. A small round 
or oval centric nucleus is observed staining a deep port-wine color, while 
many inclusion bodies (contained in vacuoles) may be seen staining a deep 
purple. Usually the entire endameba appears to be surrounded by a clear 
zone, due to retraction during drying. ^ 

11. Nasal Secretion 

This secretion seems to be of pathologic significance only in infectious 
conditions. Normally, the nasal secretion is comparatively scanty, clear, 
tenacious, odorless, salty in taste, and alkaline in reaction. It is largely 

1 Dupray (Jour. A. M. A., 1915, LXVI, 507) recommends the use of a carbolized thionin 
solution. 

^Gros, Bull. Soc. imp. nat. de Moscow, 1849, XXII, 549; Steinberg, Souremenaya 
meditsina, Dissert. Kiev, 1862; Grassi, Gazz. med. Ital.-Lomb., 1879, XXXIX, 446; Flex- 
ner, Bull. Johns Hopkins Hosp.,*i892, III, 104; Kartulis, Ztschr. f. Hyg., 1893, XIII, 9; 
Prowazek, Arbeiten a. d. Kais. Gesundheitsamte, 1904, XXI, 42; Ley den and Lowenthal, 
Charite Ann., 1905, XXIX, 3; Verdun and Bruyant, L'Echo med., du Nord, 1907, XI, 375; 
Bruyant and Pehssier, Ibid., 1909, XIII, 301; Brumpt, Precis de Parasitol., Paris, 1910; 
Barrett, Dental Cosmos, 1914, LVI, 948 and 1345; Chiavaro, Editorial in Dental Cosmos, 
1914, LVI, 1089; Austral. Jour. Dent., 1914, XVIII, 343; Dental Review, 1914, XXVIII, 
1122; Bass and Johns, New Orleans Med. and Surg. Jour., 1914, LXVII, 456 and 671; 
Smith, Middleton and Barrett, Jour. Am. Med. Assn., 1914, LXIII, 1746; Evans and Mid- 
dleton, Ibid., 1915, LXIV, 422; Bass and Johns, Ibid., 553; Howe, Dental Cosmos, 1915, 
LVII, 307; Brit. Dental Jour., 1915, XXXVI, 464; Talbot, Dental Cosmos, 1915, LVII, 
485; Jour. Am. Med. Assn., 1915, LXIV, 928; Rogers, Indian med. Gaz., 1915, I, 121; 
Stewart, Med. Record, 1915, LXXXVII, 798; Dental Cosmos, 1915, LVII, 605; Stur- 
ridge, Ibid., 780; Johns, Interstate Med. Jour., 1915, XXII, 529; Smith, Jour. Am. Med. 
Assn., 1915, LXIV, 1567; Smith and Barrett, Jour. Parasitol., 1915, I, 159; Brunelles and 
Ginsberg, New York Med. Jour., 1915, CII, 554; Williams, von Shelly and Rosenberg, 
Proc. New York Path. Soc, i9i5,XV, 334; Bass and Johns, Alveodental Pyorrhea, Saun- 
ders, Phila., 1915; Hecker, Am. Jour. Med. Sc, 1915, CXLIX, 889; Smith and Barrett, 
Jour. Parasitol, 1915, II, 54; Lynch, Jour. A. M. A., 1915, LXV, 2077; Johns, Amer. Jour. 
Trop. Dis. and Prev. j\Ied., 1916, III, 367; Sanford, Surg., Gyn., and Obs., 1916, XXII, 27; 
Price, Ibid., 37; Craig, Jour. Infect. Dis., 1916, XVIII, 220; Moody, Ibid., 1916, XIX, 515; 
Hecker, Ibid., 729; Mitchell, Culpepper and Ayer, Jour. Med. Research 1916, XXXV, 51; 
Evans, Middleton and Smith, Am. Jour. Med. Sc, 1916, CLI, 210; Nowlin, Jour. Parasitol., 
1917, III, 143 and 1917, IV, 21. 



ORAL, NASAL, AURAL, AND CONJUNCTIVAL SECRETIONS 45 

composed of mucus, showing squamous and ciliated epithelium in abund- 
ance,^ occasionally leucocytes, large numbers of bacteria and Charcot- 
Leyden and triple phosphate crystals. The bacterial content is made up of 
both pathogenic and non-pathogenic organisms. 

Pathologic Changes. 

In most acute infections the nasal secretion is at first diminished in 
amount, but soon becomes very profuse. This secretion shows the same 
appearance as does the normal fluid, but as ulceration ensues may be heavily 
loaded with pus-cells and bacteria. The chronic suppurative process 
in the nose may affect any or all of the accessory sinuses, so that we may 
have very severe conditions arising from simple ulceration. Frequently 
the ulcerative and membranous conditions spoken of above may extend 
from the mouth to the nose, so that distinct diphtheritic membranes are 
frequently found in the nasal cavities. Molds may develop to such an ex- 
tent that true pathological lesions obtain. ^ 

Rhinitis. 

At present the infectious nature of the catarrhal conditions known as 
"colds" is generally accepted, many organisms, such as micrococcus catar- 
rhalis, pneumococcus, streptococcus, influenza bacillus and others, having 
been associated in different cases with the etiology. It would seem that these 
organisms are, however, to be regarded as simply secondary invaders, in many 
cases at least, and become of importance only when the discharge is purulent. 

Tunnicliff^ has found, in the mucoid stages of acute and chronic rhinitis, a 
delicate, curved, Gram-negative bacillus, which appears to have a direct 
pathogenic association with these conditions. While this Bacillus rhinitis of 
Tunnicliff is found in only 6 per cent, of normal noses, it was present in 98 per 
cent, of cases of acute rhinitis and in 90 per cent, of cases of chronic non- 
purulent rhinitis. It is, apparently, not found at all in purulent rhinitis, the 
other organisms mentioned above being the causative factors in this latter 
condition. There is little doubt that this organism has an etiologic relation- 
ship to acute and chronic rhinitis on account of its almost constant presence 
in the nose in such cases, its general absence from the normal nose, its ability 
to produce rhinitis experimentally with recovery in pure culture, and on ac- 
count of the production, in cases of acute and chronic rhinitis and in persons 
injected with the bacillus, of specific opsonins and complement-binding bodies. 

This organism may be found by spreading a fairly large amount of the 
nasal discharge (mucus) on a glass slide, drying and staining with carbol- 
fuchsin. Often a large number of microscopic fields may have to be examined 
before the organisms are found. They vary in length from 5 to 8 microns and 
from J:3 to 3^^ micron in width. The ends are pointed or slightly rounded; 
generally the bodies are slightly curved, but may be straight, wavy or bent 

^Proskauer (BerL klin. Wchnschr., i9i5,LI, 172 and 255) has shown that some of the 
cells may be melanotic. 

2 See Tilley, Jour. LaryngoL, Rhinol. and Otol., 1915, XXX, 145. 

•^Jour. Infect. Dis., 1913, XIII, 283; Ibid., 1915, XVI, 493. See Howell, Ibid., 456; 
also, Kruse, Munch, med. Wchnschr., 19 14, LXI, 1547. 



46 DIAGNOSTIC METHODS 

over at one end. A ring and an enlargement in the form of a ball are occasion- 
ally seen at one extremity. The organism is a strict anaerobe growing at 
37°C. slowly, the colonies on slightly alkahne goat-blood agar appearing 
usually in about seven days as small, dull, round growths. The organism 
does not grow on plain agar. Specimens from cultures stain a little more 
deeply than those taken directly from the mucus, but they retain their 
Gram-negative characteristics. 

Hay Fever. 

In this condition the nasal secretion is found to be increased to a large ex- 
tent at certain times of the day and much diminished at others, depending 
upon the paroxysms of the disease. Nothing of pathological importance has 
been found, however, in the examination of the nasal secretion in this 
condition, no specific organism having been identified. 

Meningitis. 

In some cases of meningitis the cerebrospinal fluid passes into the nasal 
cavity as a result of caries of the bones of the skull. This fluid may be dis- 
tinguished by the fact that it contains practically no albumin, but does show 
the presence of a reducing substance which may or may not be sugar. This 
fluid may also contain the diplococcus intracellularis meningitidis of Weichsel- 
baum. While this organism is not found in all cases of epidemic meningitis, 
yet it is found in many, so that the nasal secretion may be of some diagnostic 
importance from this standpoint. 

In the course of glanders, leprosy, plague, pneumonia, typhoid fever, 
influenza, and many other infectious diseases, the characteristic organisms of 
these conditions may be found in the nasal secretion. Recently Goldberger 
and Anderson^ have demonstrated the presence of the unknown virus of 
measles in the mixed buccal and nasal secretions. ^ 

Occasionally concretions are found in the nose, but these rarely reach 
a large size and do not have very great pathologic significance. They are 
largely composed of vegetable fibers taken in by inhalation and cemented 
by mucus which is hardened by the deposition of lime salts. 

In the condition known as ozena, the nasal secretion and fetid crusts, 
so characteristic of this disease, are found to contain numerous organisms 
of various types, among which one observes a peculiar cocco-bacillus which 
is difl&cult of cultivation and isolation. This organism was first noted by 
Perez, but it was not until the later studies of Hofer and his associates 
that it was practically established as the etiologic factor in this disease. 
To this micro-organism has been given the name coccobacilhis fcetidus ozence 
(Perez). 3 

3 Jour. Am. Med. Assn., igii, LVII, 476 and 971; Am. Jour. Dis. Child., 1912, IV, 20. 

2 See also Lucas and Prizer, Jour. Med. Research, 1912, XXVI, 181. 

3 See Hofer, Wien. klin. Wchnschr., i9i3,XXVI, loii; also, Abel and Hallwachs, Kolle 
and Wassermann's Handbuch, 19 13, VI, 534; McGowan, Jour. Laryngol., Rhinol. and 
Otol., 1915, XXIX, 57; Guggenheim, Interstate Med. Jour., 1915, XXII, 129; Horn, Jour. 
Am. Med. Assn., 1915, LXV, 788; Horn and Victors, N. Y. Med. Jour., 1916, CIV, 1094; 
Ward, Jour. Infect. Dis., 1916, XIX, 153; Ward and Beaver, Jour. Lab. and Clin. Med., 
191 8, III, 348; Ferry and Noble, Jour. Bacteriol., 1918, III, 499. 



ORAL, NASAL, AURAL, AND CONJUNCTIVAL SECRETIONS 47 

III. The Aural Secretion 

Normally no secretions appear in the external ear, with the exception of 
that of cerumen, while the secretion of the middle ear and of the internal ear 
is normally inaccessible to examination. We find, therefore, that the chief 
importance which is attached to the clinical examination of the aural secre- 
tions, is entirely a pathological one. In catarrhal and inflammatory condi- 
tions of the external auditory canal, one finds naturally very large numbers of 
organisms, with which the disease may or may not be associated. In the 
chronic inflammatory processes of the middle ear, the more important organ- 
isms found are the pneumococcus, streptococcus pyogenes, staphylococcus 
pyogenes, bacillus pyocyaneus, the bacillus of Friedlander, the bacillus coli 
communis, the diplococcus intracellularis, the typhoid bacillus, and especially 
the diphtheria bacillus. As disease of the middle ear is so commonly asso- 
ciated with disease of the naso-pharynx, it is possible to find in the discharge 
from the ear any organism which is causing trouble either in the nose or in 
the throat. Hamilton has shown the almost constant presence of the pseudo- 
diphtheria bacillus in the discharge of the running ears following scarlet fever. 

It is not an uncommon thing to find certain inflammatory processes of para- 
sitic origin in the external auditory canal. This condition is known as 
otomycosis and is frequently caused by the aspergillus niger. Besides this 
organism, many other fungi belonging to this group have been found in the 
external ear. Among these we find the aspergillus flavus and fumigatus, the 
aspergillus nidumus, the mucor septatus, the eurotium malignum, and the 
penicillium minimum. These parasites are very readily detected by remov- 
ing a small portion of the mycotic mass and spreading it thinly on a slide. 
Add a small drop of water, apply a cover-glass, and examine under a high- 
power dry lens. For the details of structure of such organisms, bacteriological 
works should be consulted. 

Besides these fungi we occasionally find in the external auditory canal 
larvae of various insects. These larv« may develop later into the full-grown 
insect and may be removed from the ear by the movements of the animal. 
In other cases such larvae have incited inflamm.atory processes which are 
occasionally troublesome, as in the case reported by Richardson. 

IV. The Conjunctival Secretions 

Under normal conditions the secretion of the conjunctiva and of the lacri- 

lal gland concerns us only very little. It is in the course of an inflammatory 

rprocess in which these secretions maybe greatly increased and greatly changed 

py the inflammation that any clinical importance attaches to them. In in- 

lammatory conditions of the conjunctiva we find certain organisms which 

'equire identification in order that proper treatment may be instituted and 

[the proper prognosis given. ^ It is to be recalled that the pseudo-diphtheria 

1 Wilder and McCullough (Jour. Am. Med. iVssn., 1914, LXII, 1156) report a case of 
Isporotrichosis of the eye in which the organisms were observed, in direct smears, as Gram- 
Ipositive oval bodies and in cultures as typical sporothrices. 



48 DIAGNOSTIC METHODS 

bacillus is practically always found in smears made from the conjunctival 
secretion, yet it is rarely, if ever, pathogenic in this situation. 

Pathologic Changes. 

Diphtheritic Conjunctivitis. 

In cases of conjunctivitis which are traceable to infection with the diph- 
theria organism, we frequently find the formation of an extensive membrane 
which consists of epithelial cells, leucocytes, and large numbers of streptococci 
along with the diphtheria bacilli. Clinically, a membrane formation on the 
conjunctiva may arise from infection of this tissue with organisms other than 
the diphtheria bacillus; hence, it is wise in all suspicious cases to submit a 
portion of the membrane to both direct microscopic and cultural examinations. 

Infectious Conjunctivitis. 

In acute infectious conjunctivitis, various organisms have been found, 
the most common ones being the Koch- Weeks bacillus, the pneumococcus, 
and the gonococcus. Occasionally one finds the staphylococcus, strepto- 
coccus, colon bacillus, influenza bacillus, Morax-Axenfeld bacillus, the 
diphtheria bacillus, and other organisms.^ 

In some regions the Koch- Weeks bacillus is frequently found as the 
etiologic factor in acute infectious conjunctivitis, while in others it is rarely, 
if ever, observed. This is an organism of the influenza group, a small, thin, 
Gram-negative bacillus, which is, so far as known, pathogenic only for the 
human conjunctiva (see cut). It grows best on media containing a slight 
amount of human blood, especially in symbiosis with the xerosis bacillus. 
This latter organism is differentiated from the Klebs-Loffler bacillus only 
by the application of the fermentation tests spoken of under Examination 
for Diphtheria Bacilli in the Throat. 

The most common bacterial cause of chronic conjunct vitis is the bacillus 
of Morax-Axenfeld, usually seen as a diplobacillus, several groups of which 
may at times be arranged in chains (see Plate V). It is a Gram-negative or- 
ganism and grows well onLoffler's blood-agar, which it digests, forming on the 
surface at the beginning of its growth, very characteristic small pits. 

Gonorrheal Conjunctivitis. 

This form of conjunctivitis is much more common than is generally sup- 
posed, so that the identification of the organism is of great importance. In 
the development of this type of conjunctivitis, large or small amounts of pus 
are invariably present between the folds of the conjunctiva. A portion of this 
pus may be collected by means of a cotton swab or a platinum loop and 
smeared thinly over a slide. This smear is then fixed in the flame and is 
stained for the gonococcus by both the methylene blue and Gram stains. 
The technic of this latter staining process, as well as the characteristic 
appearance of the organism, will be discussed in a later section. 

^See Axenfeld, Kolle and Wassermann's Handbuch, 1913, VI, 545, 572 and 587; also, 
Noguchi and Cohen, Jour. Exper. Med., 1915, XXII, 304; Irons, Brown and Nadler, Jour. 
Infect. I)is., 1916, XVIII, 315. 



PLATE VI. 






:'^^-^ 



^ ,-•' <r 



^ 

■•'f: 



Kathapfh« Hill 



MORAX-AXENFELD DiPLOBACILLUS. (GrAM's StAIN.) 

Courtesy of Dr. Brown Pusey. 



P^ Kathar.r^ H ill 



Koch- Weeks Bacillus. (Gram's Stain.) 
Courtesy of Dr. Brown Pusey. 



PLATE VII. 






XathoritT.€ HJit 



Trachoma Bodies or Prowazek-Geeeff. (Giemsa Stain.) 
Courtesy of Dr. Brown Puse3\ 



ORAL, NASAL, AURAL, AND CONJUNCTIVAL SECRETIONS 49 

Trachoma. 

Lately, a great deal of attention has been given to some bodies, which are 
found in trachoma and which are, possibly, the long-sought cause of this infec- 
tious disease of the conjunctiva. These organisms are known as the Prowa- 
zek-Greeff bodies and are shown in the accompanying cut. They are best 
stained by the Giemsa stain (see Blood), smears being made in the usual 
manner. 

The present status of these bodies is that they are almost always found 
in the acute stages of this disease and are, occasionally, observed in other 
conditions which do not present the clinical features of trachoma. Noguchi 
and Cohen (Jour. Exper. Med., 1913, XVIII, 572) report the cultivation of an 
organism, resembling the trachoma body, from cases of trachoma with and 
without cell inclusions. Whether or not they are the true etiologic factor 
must still be considered an undecided question.^ 

Vernal Conjunctivitis. 

An interesting point in ithe diagnosis of the above condition, as well as 
of the conjunctivitis of hay fever, is the fact, first observed by Herbert^ and 
confirmed by many, that in these two types of conjunctivitis eosinophile poly- 
morphonuclear leucocytes are found in abundance, whereas in the ordinary 
forms of conjunctivitis such cells do not obtain. Pusey^ has, therefore, been 
led to believe that these facts point strongly to a similar etiologic factor, 
namely pollen, in the vernal conjunctivitis and in the conjunctivitis of hay 
fever. 

BIBLIOGRAPHY 

1. AxENFELD. Die Bakteriologie in der Aiigenheilkunde. Jena, 1907. The Bac- 

teriology of the eye. New York, 1908. 

2. Davis. Les Mikrobes de la Bouche. Paris, 1890. 

3. Kohl. Die Heferpilze. Leipzig, 1908. 

4. Litchfield. Diphtheria in Practice. London, 1908. 

5. Mallory and Wright. Pathological Technic. Philadelphia, 19 15. 

6. Miller. Die Mikro-organismen der Mundhohle. Leipzig, 1889. 

7. NuTTALL AND Smith. Bacteriology of Diphtheria. Cambridge, 1908. 

8. Sticker. Die Bedeutung des Mundspeichels in physiologischen und patho- 

logischen Zustanden. BerHn, 1899. 

1 See report from the Laboratories of the New York Health Department found in Proc. 
New York Path. Soc, 191 2, XH, 17; Williams and associates, Jour. Infect. Dis., 1914, XIV, 
261. 

2 Brit. Med. Jour., 1903, II, 73; Bollack, Presse Med., 191 7, XXV, 3. 

3 Jour. Am. Med. Assn., igiijLVII, 1207. 



CHAPTER III 
GASTRIC CONTENTS 

I. General Considerations 

The gastric juice is the product of secretory activity of the glands of the 
stomach. Different series of glands contribute separate elements to the 
secretion, so that we find much variation, under pathologic conditions, in the 
composition of this fluid. 

The stomach should be regarded as a dilated and specialized portion of the 
general digestive tube, its walls consisting of the following four coats: mucous, 
submucous, muscular, and fibrous. From the standpoint of secretory activity 
the internal or mucous coat is the most important. This mucous membrane 
is covered throughout its entire length by a single layer of simple columnar 
epithelium. It follows the various folds or rugcB dipping down in places to line 
the orifices and ducts of the tubular glands which are of such importance in the 
digestive activity of the stomach. The gastric glands are of two kinds, the 
peptic or fundus glands, situated in the middle and cardiac thirds of the 
stomach, and the pyloric glands, found in the pyloric third of the stomach. 

Peptic Glands. 

These glands are slightly wavy simple tubular depressions, in which a duct, 
a neck, and a fundus are recognizable. In exceptional cases the fundus is 
divided, while in nearly all it is tortuous or spiral its extremity being often 
sharply bent at right angles to the general axis of the tube (Piersol) . In these 
peptic glands are found two types of cell. The first, known as the central, 
chief or adelomorphous cells, bound the lumen of the gland and form the bulk 
of the glandular epithelium. These cells are either polyhedral or columnar in 
form and each contains a spherical nucleus situated within the granular pro- 
toplasm. These cells do not stain readily with aniline dyes. The chief func- 
tion of these central cells of the peptic glands is to secrete the rennin and lipase 
which are present in the gastric juice. The second type of cell in the peptic 
gland is known as the parietal, acid or oxyntic cell and is situated in the 
periphery of the gland immediately below the basement membrane. These 
cells are more oval or angular in form, are larger than the chief cells, are more 
finely granular in structure and stain deeply with the aniline dyes. 

The Pyloric Glands. 

These glands are characterized by their relatively long wide ducts into 
which the several divisions of the body open; the tubular compartments are 
wavy and tortuous and frequently end in slightly expanded extremities. The 
duct is Uned by tall columnar epithelium, the cells becoming lower and broader 
as they approach the neck and toward the fundus. The cells contain finely 

so 



GASTRIC CONTENTS 5 1 

granular protoplasm and do not secrete mucus but a thin albuminos liquid. 
Parietal or acid cells do not occur in the pyloric gland, being confined to 
the true peptic gland (Piersol). 

It will thus be seen that the active portions of the gastric juice are secreted 
by the fundus glands, the pyloric glands contributing nothing except a small 
amount of the ferments and liquid portion, the mucus being largely derived 
from the goblet cells which line the entire stomach and the wider portion of 
the glandular ducts. These ferments do not exist in the cells as such, but 
rather in the form of zymogens or prozymogens which become active only 
in the presence of the free hydrochloric acid.^ 

The free hydrochloric acid of the gastric juice is formed in the parietal cells 
of the peptic gland. ^ The mechanism of this formation is not absolutely estab- 
lished, but it seems probable that this free acid arises from the chlorids taken 
up from the blood by these cells. Just what is the active agent in causing the 
conversion of the chlorids into free acid seems to be in doubt, but it may be 
either the continuous action of carbonic acid or, as Maly assumes, the inter- 
action of the sodium phosphate (Na2HP04) with the chlorids of the cell. 
It is also probable that the osmotic influences may be very great in the pro- 
duction of this free hydrochloric acid as Koeppe advocates. This acid is 
present at all times in the normal stomach, being found even in cases of 
extreme starvation. ' 

The careful work of Pawlow^ has shown that various factors influence the 
quantity and quality of the normal gastric juice. He asserts that the " appe- 
tite is the first and mightiest exciter of the secretory nerves of the stomach, 
a factor which embodies in itself a something capable of impelling the empty 
stomach of the dog in the sham feeding experiment to secrete large quantities 
of the strongest juice. A good appetite in eating is equivalent from the outset 
to a vigorous secretion of the strongest juice; where there is no appetite this 
juice is also absent." Moreover, under natural conditions, the stimulation of 
food is a very important factor. The administration of a diet causes a secre- 
tion of gastric juice which is directly proportionate, both in amount and 
activity, to the diet taken. We find, according to Chigin, that the greatest 
digestive power is shown by the juice excreted after the administration of 
bread, although the total acidity is greatest following an intake of meat. 
If we compare equivalent weights of food material we find that flesh requires 
the most gastric juice and milk the least; but taking equivalents of nitrogen, 
bread needs the most and flesh the least. In this connection it is well to 
remember that the gastric secretion varies from hour to hour. Thus the 
most active juice occurs with flesh in the first hour, with bread in the second 
and third hour, and with milk in the fifth to the sixth hour. The point of 

^A secretagogue, called by Edkins gastrin, is believed to be formed by contact of certain 
substances with the gastric mucosa. This gastrin apparently causes a true gastric secretion 
(see Keeton and Koch, Am. Jour. Physiol., 1915, XXXVH, 481). 

2 See Harvey and Bensley, Biol. Bull., 191 2, XXIII, 225; also Lopez-Suarez, Biochem. 
Ztschr., 1912, XLVI, 490; Hammett, Anat. Rec, 1915, IX, 21. Bergeim, Proc. Soc. Exper. 
Biol, and Med., 1914, XII, 21. 

^The Work of the Digestive Glands. London, 1902. See, also, Wolfsberg, Ztschr. f. 
physiol. Chem. 1914, XCI, 344. 



52 



DIAGNOSTIC METHODS 




all this is that the rate and time of secretion of the gastric juice is always 
characteristic for each diet. 

Moreover, it has been found that the hydrochloric acid first secreted com- 
bines at once with the proteins of the various food stuffs, so that we may find 
no free hydrochloric acid in the gastric contents, although the secretion may 
be normal and may show a very high degree of total acidity. 

II. Methods of Obtaining the Gastric Contents 
Unless the patient is one who can easily eject the contents of the stomach 
by vomiting, it is necessary to resort to the introduction of the so-called 

stomach- tube for the removal of the contents. 
This stomach-tube consists of a long, soft rubber 
tube about 75 cm. in length, having a lumen 6 to 
7 mm. in diameter and provided with either two 
oval lateral openings or with three, one being at 
the end of the tube.^ Before introduction of the 
stomach-tube, it should be moistened with warm 
water and should be thoroughly cleaned. 
Introduction of the Tube. 

The patient must be in a sitting posture, a 
towel or a rubber sheet being placed about his 
neck to prevent soiling of the clothes with the 
saliva or material which is occasionally 
brought up during the passage of the tube. 
False teeth should be removed and anything interfering with the passage of 
the tube should be avoided. In patients who are hypersensitive, a lo per 
cent, solution of cocain is applied to the pharynx. The head of the patient is 
now bent slightly forward, never backward as some advise, and the mouth 
slightly opened, care being taken never to use a depressor on the tongue. 
The rubber tube, held as one would a pen, is passed gently backward over 
the tongue until its tip strikes the posterior wall of the pharynx, when it runs 
downward and may be readily introduced into the stomach, by slight forcing. 
As the tube reaches the esophagus, many patients complain of a sense of suffo- 
cation, which is not real but apparent. The tube interferes in no way with 
the normal respiratory movements and hence the patient should be cautioned 
to breathe normally and not forget to breathe. If the patient will swallow 
normally, the passage of the tube is greatly facilitated. It occasionally hap- 
pens that highly nervous patients have great difficulty in swallowing this 

^Rehfuss (Am. Jour. Med. Sc, 1914, C XL VII, 848) has devised a special stomach tube 
with a special tip, slotted with large perforations. This tip, on account of its weight, passes 
to the most dependent parts of the stomach and permits of more certain withdrawal of the 
stomach contents as well as a fractional withdrawal and examination of the gastric contents 
which is the most exact and satisfactory method of study. See Schiitz, Arch. f. Verdauung- 
skr., 1914, XX, 304; Lauza, Riforma Med., 1915, XXXI, 561; Rehfuss, Bergeim and Hawk, 
Jour. A. M. A., 1914, LXIII, 909; Rehfuss, Ibid., 1915, LXIV, 569; Clark and Rehfuss, 
Ibid., 1737; Fowler, Rehfuss and Hawk, Ibid.. 1915, LXV, 1021; Talbot, Ibid., 1916, LXVI, 
1849; Best, Ibid., i9i6,LXVII, 1083; Fishbaugh, Ibid., 1275; Wilensky, Ibid., i9i7,LXVIII, 
891; Crohn and Reiss, Am. Jour. Med. Sc, 191 7, CLIV, 857; Robinson, Arch. Int. Med., 
191 7, XIX, 220; Fowler, Jour., Biol. Chem., 191 7, XXXII, 389; Cessna and Fowler, Ibid., 
1919, XXXIX, 25. 



Fig. 14. — Stomach tube. 



GASTRIC CONTENTS 53 

tube, so that it may be necessary to defer the withdrawal of the contents to a 
second or even a third period. It is never wise to excite a patient by forcing 
matters at any stage of the investigation. If any sign of cyanosis or marked 
pallor is evident the tube should be immediately withdrawn and a second at- 
tempt made at some later time. When the tube has reached the floor of the 
stomach, which is in normal cases about 40 cm. from the incisor teeth, a dis- 
tinct resistance to further passage of the tube will be noticed. This point 
should be carefully observed as the forcing of the tube beyond this point may 
produce rupture of the stomach wall or may cause the tube to " buckle."^ In 
this latter condition it will be impossible to withdraw the stomach contents. 
Many of the tubes used for gastric examination have a mark indicating the 
normal length of tube from the incisor teeth to the stomach wall, so that one 




-Turck's aspiration apparatus. 

has a definite idea when he has introduced the tube into the right point. In 
some cases the gastric juice will commence to flow from the tube as soon as it is 
properly introduced, but in the majority of cases some help is necessary to 
start the siphonage. Frequently all that is needed is to ask the patient to 
bear down with his abdominal muscles or to cough a little. In other cases 
aspiration is necessary. One may employ an ordinary Politzer bag or a Boas 
bulb for starting the fluid in the tube. This is very readily done by compress- 
ing the bulb and applying it, while CQ.mpressed, to the end of the tube in such a 
way that the suction will be sufficient to draw the material into the tube. 
Once started, the material flows quite readily, but it may be necessary to use 
aspiration several times as the tube may become clogged. 

If it is desired to wash out the stomach, either to obtain the total gastric 
contents or for the purpose of mere lavage, a funnel is attached to the external 
end of the stomach-tube and about 500 c.c. of water are allowed to flow 
through the tube into the stomach. In this operation the funnel is held 
either on a level with the patient's mouth or a very Httle bit above. By 
depressing and inverting the funnel over a suitable vessel, before all the water 
has left it, return flow will soon set in and the stomach will be practically 
emptied by siphonage. In some cases it becomes necessary to add more 

^In this connection see Harmer and Dodd, Arch. Int. Med., 1913, XII, 488. 



54 DIAGNOSTIC METHODS 

water, but in no case should any be added after the patient complains of a 
feeling of distress. 

In collecting the stomach contents one should avoid as far as possible any 
admixture with the saliva which is more freely excreted at this time than 
normally. This is best done by wrapping a cloth about the tube so that the 
material may be absorbed as it runs along the side of the tube. After one has 
obtained the gastric contents, the tube is compressed with the fingers and is 
rapidly withdrawn, care being taken to keep up the compression so as to hold 
in the tube material which has not already passed into the receiving vessel. 

In cases in which water has been introduced to wash out the stomach after 
the gastric contents have been obtained, one should be careful to note the 
amount of fluid poured into the stomach so that he may be able to judge of 
the amount again received. 

Not every case with which the practitioner meets is amenable to such 
manipulation. We find as especial contraindications uncompensated 
valvular lesions of the heart, arteriosclerosis, aneurysm, advanced pulmon- 
ary tuberculosis, marked emphysema, acute febrile diseases, severe hemor- 
rhage, especially from ulcer or carcinoma, and excessively developed nervous 
antipathy. 
Test Meals. 

As the secretion of the gastric juice is so dependent upon administration 
of food, it has become the custom to use certain combinations of food principles, 
which will excite gastric activity and enable us to obtain a juice which wdll 
give us more or less definite ideas of its composition in the condition investi- 
gated.^ It must be remembered that marked idiosyncrasy toward certain 
foods exists, so that we may not use in all cases the same sort of a diet for ex- 
citing the gastric juice. The results obtained in pathologic conditions are 
compared with those obtained from normal individuals under the influence 
of the same diet. In this way we are able to say, with some degree of cer- 
tainty, that a suspected case shows normal or abnormal gastric relations. 
These diets, the so-called test meals, are always given to the fasting stomach 
and are removed after a suitable time by the use of the stomach-tube. The 
time best suited for the administration of these meals is in the morning, as the 
stomach has had occasion during the night to empty itself of most of its 
contents. (See footnote on p. 52 for references regarding the fractional 
method of gastric analysis.) 

Ewald Test Meal. 

This meal, which is, perhaps, the most frequently eniployed in general 
work, consists of a roll or piece of bread or toast without butter and two cups 
of water or tea^ without milk or sugar. In approximate figures this will repre- 

^See Smithies, Am. Jour. Med. Sc, 1915, CXLIX, 183; also, Heaton, Brit. Med. Jour. 
1915, I, 710. 

^Kober, Lyle and Marshall (Jour. Biol. Chem., 1910, VIII, 95) have shown that it is 
almost impossible to detect blood in the presence of tannic and gallic acids, as found in tea. 
It is wise, therefore, not to use tea in the test meal when blood is suspected. Bergeim, Reh- 
fuss and Hawk, Jour. Biol. Chem., 1914, XIX, 345, show the direct stimulatory power of 
water. Brendle (Med. Klin., 1916, XII, 1257) advocates a test meal of 100 c.c. of water. 



GASTRIC CONTENTS 55 

sent 35 grams of wheat bread and 400 ex. of water or tea.^ The bread should 
be well masticated so that the later withdrawal of the contents may not be 
interfered with by the plugging up of the openings in the tube. The contents 
are removed one hour later and consist normally of 30 to 50 c.c, depending 
both upon the skill of the operator and upon the condition of the stomach. 
Hypermotility of the stomach will diminish the quantity of contents received, 
while a hypomotility will increase the quantity. 

Boas Test Meal. 

This meal consists of a dish of oatmeal prepared by concentrating to 500 
c.c. a liter of water to which a tablespoonful of oatmeal is added. This 
meal was advised to prevent the introduction into the stomach of laqtic acid 
which is a normal constituent of bread. While this small amount of lactic 
acid introduced in the Ewald meal has little significance, yet in doubtful cases 
it is well to avoid it. The contents of the stomach are withdrawn one hour 
later when the amount may be very small. If the stomach shows normal 
digestive powers most of the material will be then passed into the intestine, 
while an appreciable amount of material would indicate either a dilatation of 
the stomach or pyloric obstruction. 

Riegel Test Meal. 

The test meal has the advantage of permitting the patient to use a diet 
which is more normal than either of the ones previously mentioned. This 
diet is more important in America, where we are not accustomed to the con- 
tinental breakfasts, than it is in Germany where the Ewald and Boas meals 
are more usual. 

The Riegel meal is given in the middle of the day at a time when the 
patient is accustomed to such a meal. It consists of about 400 c.c. of soup, 
200 grams of beef -steak, and either two slices of white bread or 150 grams of 
mashed potato along with one glass of water. This meal is withdrawn at the 
end of three to four hours. It has the advantage of allowing us to judge of the 
length of time which the food remains in the stomach under normal conditions 
and, also, to form an opinion of the rate and amount of digestion which has 
taken place. This meal incites a more nearly normal gastric juice than does 
the Ewald or Boas meal, but it is such that clogging of the stomach tube by 
particles of undigested food frequently occurs. 

Fischer Test Meal. 

This meal, introduced by an American physician, has the advantage of 
more nearly approaching an American breakfast than the others. It consists 
of the bread and tea of the Ewald meal along with a quarter of a pound of 
finely chopped lean beef broiled and seasoned. The contents are removed 
at the end of three hours. 

^Roberts (Jour. Am. Med. Assn., 191 2, LVIII, 753) adds to this breakfast 30 grams of 
lactose. He extracts the meal as usual and then washes out the stomach with 200 c.c. of 
water. The total gastric juice excreted and the acidity when excreted are calculated by the 
method of Mathieu (p. 58). 



56 DIAGNOSTIC METHODS 

Salzer Test Meal. 

This is in reality a double meal and is given as follows: For breakfast 
the patient receives 30 grams of lean cold roast meat, finely chopped, 250 c.c. 
of milk, 60 grams of rice, and one soft-boiled egg. Four hours thereafter a 
second meal is given, consisting of 35 to 70 grams of stale wheat bread and 400 
c.c. of water. The contents are then rejnoved one hour after this second 
meal. Under normal conditions of digestion and motility the stomach con- 
tents should show no remnants of the first meal. 

Sahli Test Meal. 

This meal was introduced to enable the worker to examine quantitatively 
the material withdrawn. The inconstant composition of the ordinary test 
meals makes it rather difficult to judge of the digestive power of the stomach. 
Sahli has introduced, therefore, a soup prepared as follows: Twenty-five 
grams of flour and 15 grams of butter are placed in a pan and browned over a 
fire. Three-hundred and fifty c.c. of water are then added and the whole 
boiled for five minutes (the loss in volume being replaced by fresh water) after 
which it is seasoned with a little salt. In this soup the fat is in the form of a 
very fine emulsion and the taste is so pleasant that a more nearly normal 
stimulus to gastric secretion is offered. The patient is now allowed to take 
300 c.c. of this soup, while the remaining 50 c.c. are retained for a determi- 
nation of the fat content. The contents are withdrawn, one hour after the 
meal, from the stomach which must have been thoroughly washed out prior 
to the administration of the meal.^ 

We then determine the absolute amount of fat remaining in the stomach 
after the test digestion and compare this amount with that introduced. As 
we cannot be sure that the entire stomach contents have been withdrawn, we 
must know the residual amount of gastric juice. For this purpose one resorts 
to the method of Mathieu, which will be discussed later (p. 58). 

The amount of fat both in the original soup and in the withdrawn stom- 
ach contents is then determined and the total gastric juice calculated. This 
method of fat determination will be given in detail under Milk, to which 
the reader is referred. 

Calculation of Results. 

"The following calculations are possible from a consideration of the resi- 
due from the acidity of the gastric filtrate, and from the difference between the 
amount of fat found in the ingested flour soup and that found in the expressed 
contents. 

''By the addition of the value X, found in the calculation of the residue 
(p. 58), to the amount of contents expressed after one hour, there is obtained 
the volume of the contents which were actually present in the stomach at the 
end of that period. This we designate as To. From the absolute fat-content 
of To, there can be determined how much of the volume can be ascribed to the 
ingested flour soup. The amount of fat remaining in the stomach serves, 

^ Sahli (Lehrbuch d. klin. Untersuchungsmethoden 6 Aufl., 1913, I, 656) apparently now 
prefers a soup prepared from Maggi or Teston bouillon and yolk of egg instead of the above. 



I 



GASTRIC CONTENTS 57 

therefore, as a measure for the amount of soup remaining. This is designated 
as Su. Representing this mathematically, we have the proportion To : Su: 
F: f, in which F represents the percentage fat content of the soup and f that of 
the expressed contents. To — Su will give, of course, the volume of gastric 
juice in the expressed contents. If the acid-content of To has been deter- 
mined, it is possible from these data to proceed further and to calculate what 
acidity was possessed by the pure gastric juice as it was excreted. Thus if 
75 c.c. of pure gastric juice are present in the stomach contents, whose volume 
amounts to 150 c.c. with 2 per cent, acidity, then the acid-content of the pure 
gastric juice is evidently 4 per cent. The determination of this acidity may 
be expressed by the proportion. To : A : : Ma : a, in which To represents the 
amount of expressed contents including the residue. Ma the amount of secre- 
tion contained in the expressed contents (To — Su) , A equals the acidity of the 
pure secretion in per cent, and a the percentage acidity of the expressed con- 
tents" (Sahli). 

It is important to remember in selecting a test meal for any given case that 
the tastes of no two persons are alike and that no two persons will react identi- 
cally toward the stimulation of the same test meal. The results can only be 
comparative and have, in this sense, some value. Too much ridigity in ad- 
ministration of such meals wiU lead frequently to mistakes in diagnosis, so 
that one should learn to vary his test meals rather than to rely upon a single 
one in all cases. Another point to be borne in mind is that the meals should 
be removed at the time of optimum secretion, which may not in all cases be at 
the end of one hour with the Ewald meal.^ "The technic commonly em- 
ployed for estimating gastric function is entirely inadequate, inasmuch as it 
indicates but one phase in a constantly changing cycle, and that phase is by 
no means always the high point in the digestive curve" (Rehfuss, Bergeim and 
Hawk). 

III. Macroscopic Examination 

' The gastric juice is a clear, colorless, easily filtered, levorotatory fluid hav- 
ing a distinctly acid reaction, an acid taste, and a characteristic odor. Its 
specific gravity, when the stomach is empty, ranges between 1004 and 1006.5; 
after the ingestion of food from loio to 1020 and more than 1020 when the 
production of acid is diminished (Landois). Its cryoscopic point is —0.38 
degree to —0.444 (Roth and Strauss). 

Amount. 

The figures for the total amount of gastric juice secreted in 24 hours are 
variable. Carlson^ shows that an adult secretes a total of about 1 500 c.c. in 24 
hours, appetite being a potent factor in this process. The amount of fluid ob- 
tained one hour after an Ewald meal is from 20 to 50 c.c, although larger 
amounts ranging from 200 to 500 c.c. indicate either diminished motility or 
hypersecretion, on the one hand, or dflatation associated with pyloric 
stenosis, on the other. It is to be remembered that the quantity of juice 

^ See Ettinger, Internat. Beitr. z. Ernahrungsstor., 1913, IV, 454. Frenkel-Tissot, Cor.- 
Bl. f. Schw. Aerzte, 1919, XLIX, 1423. 
2 Am. Jour. Physio]., 1915, XXXVII, 50. 



58 DIAGNOSTIC METHODS 

secreted is influenced by the appetite and by the amount and character of 
the food taken, as well as by the age and sex of the patient and the time of 
day at which the food is taken. The largest amounts of gastric juice are 
found in cases of hypersecretion when it is not uncommon to find a liter or 
more of gastric juice in the non-digesting stomach. 

In order to determine the total amount of gastric juice secreted, one can- 
not rely upon the quantity removed as there is always a slight residuum. 
The method of Mathieu and Remond is commonly used for such determina- 
tions. This gives results very nearly exact, at least for clinical purposes. 
With this method the gastric contents are removed, as nearly as possible, after 
an Ewald meal. A definite quantity of water, usually 300 c.c, is then poured 
into the stomach through the tube and is thoroughly mixed by moving the 
funnel up and down and by pressure upon the stomach. As much as possible 
of this added fluid and the remaining gastric juice is removed and collected in 
a separate vessel. The acidity of the undiluted as well as of the diluted stom- 
ach contents is then determined by titration. From the difference in these 
two values conclusions may be drawn as to the degree of dilution and to the 
residual amount of stomach contents which was not expressed. The amount 
expressed plus the residual amount equals the total gastric contents. 

The following is the method of calculation according to Mathieu: 

Let a = acidity of the undiluted gastric contents. 
Let b = acidity of the diluted gastric contents. 

Let x = amount of the test meal remaining in the stomach after ex- 
pression. 

Let 300 c.c. = the amount of water introduced into the stomach for dilu- 
tion. Then 

a : b : : x -{- 300 : x 
ax = b (x -|- 300) 
300 b 



X = 



a — b 



An absolutely accurate result, in the study of gastric activity, can be ob- 
tained only when the total quantity of gastric juice is known. ^ It is, there- 
fore, necessary in stating, for instance, the acidity of a stomach contents to 
calculate the total available acidity rather than the mere degree of acidity. 
It is self-evident that a stomach contents expressed in the ordinary way, 
which shows an acidity of 40°, may have this acidity in a total quantity of 50 
c.c, while an acidity of 40°, with a total quantity of 200 c.c, would represent 
actually four times as much hydrochloric acid available. It would seem, 
therefore, to the writer that the method of representing acidity in terms of de- 
grees without any reference to the amount of gastric contents is absolutely 
irrational. 
Color. 

Gastric juice is normally a practically colorless liquid, although at times 
it may be somewhat opaque and, therefore, much whiter in color. Variations 
^See Rehfuss, Jour. Am. Med. Assn., 1915, LXIV, 569. 



GASTRIC CONTENTS 59 

in this colorless fluid are observed after test meals due to admixture of various 
food products, so that we may have distinctly brownish colorations due to the 
tea or particles of toasted bread, while in the test meals consisting of meat the 
color may be more of a reddish tone. 

Pathologically, we may find a distinct red color due to the presence of 
blood. This bright red color comes from the presence of fresh blood from a 
hemorrhagic gastric ulcer or may be derived from abrasions of other portions 
of the alimentary tract. If the blood has been thoroughly mixed with the 
stomach contents for some time it may appear in the form of a brownish-black 
deposit, the so-called coffee-ground material. The blood in these cases is in 
the form of hematin and must be tested for as later outlined. This coffee- 
ground appearance is particularly evident in cases of gastric carcinoma. 

The color of the gastric contents may be either a yellow or a green, due to 
the presence of bilirubin in the former case and biliverdin in the latter. This 
biliary pigment should be detected by the tests outlined under Urine. The 
presence of bile in the gastric contents is indicative of duodenal occlusion. 

In cases of intestinal occlusion below the duodenum we occasionally find 
fecal matter in the gastric contents. This is characterized by the brownish- 
black coloration and by its intense odor. 

Odor. 

The normal gastric juice is practically odorless or very slightly sour. Ad- 
mixtures of material coming from the intestines cause a very intense odor, 
while the material rising from abscesses along the alimentary tract above the 
stomach will frequently give rise to a very offensive odor. In the vomitus ob- 
tained under various pathological conditions the odor may be very character- 
istic. Thus, in uremia we may find a distinct odor of ammonia, in alcohol 
intoxication a distinct alcoholic odor is evident, while in cases of stagnation of 
gastric contents an intensely strong odor is observed. In cases of dilatation 
we frequently find the organic acids so much increased in amount that dis- 
tinct odors are noticeable. 

Consistency. 

The normal stomach contents are usually watery in character, but may 
vary due to admixture with extraneous material. After test meals or follow- 
ing a vomiting spell we may find portions of unchanged protein or carbohy- 
drate material. The amount of bread taken with an Ewald meal should be so 
far digested in one hour as to form a puree-like mass which settles out on 
standing. Various food residues are, of course, present in the vomitus so that 
the consistency and appearance may give us much valuable information re- 
garding the digestive process. In cases of mucous catarrh or in those show- 
ing either a diminution or an increase in the amount of hydrochloric acid we 
may find after a test meal the presence of large amount of tough, slimy, mu- 
coid material, which may be so abundant as practically to make it impossible 
to filter the contents. The consistency of such material may be almost that 
of a paste, or may be simply that of a thick syrup which on pouring from the 
vessel onto the filter will form distinct mucoid threads. The presence of an 



6o DIAGNOSTIC METHODS 

increased amount of mucus is of some diagnostic importance and should,, 
therefore, be looked for under all circumstances. 

Gastric Contents from Fasting Stomach. 

The stomach is practically never empty, always containing a certain 
amount of acid fluid. ^ Boas considers anything between lo and loo c.c. as a 
normal amount of material for the fasting stomach. Anything above this 
amount would mean either motor insufficiency or hypersecretion. One may 
differentiate these two conditions by washing out the stomach at night, when 
the material withdrawn in the morning will be extremely scanty if the condi- 
tion is one of motor insufficiency. 

This fluid from the fasting stomach is thin, has a specific gravity of 1004 
to 1005, contains some free hydrochloric acid, no lactic acid, and no bacteria. 
It is very commonly bile-stained, may be alkaline from the presence of pan- 
creatic juice and may contain large amounts of mucus. As such material is 
always found in the fasting stomach it is well to make it a rule to wash out the 
stomach the night before giving a test meal. 

Vomitus. 

In those cases which are associated with frequent vomiting we may obtain 
much valuable information from the examination of the ejected material. 
The amount of material vomited will depend, of course, upon the motility of 
the stomach. In cases of dilatation or of stenosis we frequently find two or 
three quarts of material, while in conditions associated with hypermotility we 
may have simply a scanty highly mucoid vomitus. The presence of food par- 
ticles will give much information as to the digestive power of the stomach. If 
undigested meat fibers are found in the vomitus, ejected three hours after eat- 
ing, one may assume more or less disturbance of protein digestion. If parti- 
cles of unchanged bread are found, three hours after taking, the disturbance in 
protein digestion is probably more marked than in the case of the meat fibers. 
If an individual vomits bits of food more than seven hours after a meal, some 
impairment of motility must exist, according to Sahli, for after that interval 
even a hearty meal should have completely left the stomach. The vomiting 
of an acid liquid containing no food particles is quite characteristic of hyper- 
secretion of gastric juice. 

The degree of acidity of the vomitus as well as the amount of hydrochloric 
acid present very frequently enables us to judge of the activity of the juice. 
These figures will not be as reliable as are the ones obtained after a test meal, 
but may serve in cases in which the stomach-tube cannot be passed. 

Frequently one finds a vomitus which is quite foamy and smells strongly of 
the volatile fatty acids. In such conditions we may assume a diminution in 
the amount of hydrochloric acid, which normally prevents the occurrence of 
any such decomposition, or we may ascribe this condition to simple stagna- 
tion of the gastric contents. Such contents will show microscopically the 

1 See Carlson, Am. Jour. Physiol., 1915, XXXVII, 50; also, Rehfuss, Bergeim andHawk? 
Jour. Am. Med. Assn., 1914, LXIII, 11 and 909; Fowler, Rehfuss and Hawk, Ibid., 1915, 
LXV, 1021; Fowler and Zentmire, Ibid., 1917, LXVIII, 167; Raymond and Robert, Bull, 
soc. med. hospitaux de Paris, 191 8, XLII, 1134. 



GASTRIC CONTENTS 6 1 

presence of large numbers of sarcinae ventriculi, yeast fungi, and various 
bacteria. 

The blood in the vomitus varies from a slight streaking of the material to a 
fluid which shows intimate mixing with the gastric contents. In cases of re- 
cent hemorrhage, which is particularly common in ulcer of the stomach, an 
abundant admixture of fresh arterial blood or of dark coagulated blood is 
observed. Brown or black cojffee-ground-like material is particularly sug- 
gestive of carcinoma, although the same condition may result from erosion 
of the gastric mucous membrane when associated with hyperacidity or 
hypersecretion. 

An admixture of bile, producing a yellowish or greenish discoloration, may 
occur with any type of vomiting, but more especially from an empty stomach 
and in that associated with duodenal obstruction. A biliary vomiting is 
frequently observed in peritonitis and may be due to the fact that there is no 
counterpressure from the gastric contents to prevent regurgitation from the 
duodenum. This green vomitus may not always be due to the presence of 
bile, but may come from contamination with various chlorophyll-containing 
organisms. 

Almost all types of vomitus contain mucus. In some cases we find abun- 
dant tough, slimy masses which seem to be indicative of mucous catarrh of 
the stomach or of a diminution in the amount of hydrochloric acid. 

Fecal vomiting is a sign either of complete motor insufficiency of the intes- 
tine as found at times in peritonitis, or indicates intestinal obstruction, either 
in the lower part of the small intestine or in the large bowel. The brownish- 
black color of this vomitus and the distinct odor render it very characteristic, 

Asiatic cholera and cholera nostras are associated with a vomitus which 
is abundant, alkaline in reaction, contains white flakes of mucus and epithelial 
cells, and large numbers of bacteria, both Koch's spirillum and the Finkler- 
Prior spirillum and various other unidentified types. This vomitus of cholera 
is known as the ^^ rice-water ^^ vomitus. 

The time at which vomiting occurs is frequently of great importance from 
the diagnostic standpoint. If it be at the height of digestion and during in- 
tense pain the condition is probably one of ulcer. If during or shortly after 
eating we may have either gastritis, a neurosis, or cancer. If it is frequent in 
the morning before breakfast and seems to be independent of eating the con- 
dition is probably one of dilatation. While these statements are not infallible, 
yet they are applicable in the majority of cases. 
Gastric Contents After Test Meals. 

The amount of material obtained after a test meal has some diagnostic 
importance. As previously stated, one obtains after an Ewald or Boas meal 
from 20 to 50 c.c. of contents, but these figures may vary to as high as 500 c.c. 
Hypersecretion or motor insufficiency are the chief causes of such increased 
amounts, the former being more probable if a large amount of free hydro- 
chloric acid is present along with the excessive amount of fluid. ^ A larger 

^ vanSpanje (Nederl. Tijdschr. v. Geneesk., 1913, LVIII, 213) calls attention to the dry 
test meal of triscuit in estimating the motor efficiency of the stomach. 



62 DIAGNOSTIC METHODS 

proportion of solid undigested material is observed in cases of pure motor 
insufficiency, but we frequently have a combination of both conditions. Ab- 
solute proof of the diminished motility is found in the presence of more than 
a trace of food in the stomach seven to eight hours after a meal. The general 
appearances of the material obtained after a test meal are those previously 
discussed. 

IV. Microscopic Examination 

The microscopical examination of the gastric contents is usually made on 
material withdrawn from the stomach after test meals, but the vomitus is oc- 
casionally examined. The gastric juice is practically never free from rem- 
nants of food, such as meat threads or starch granules, although nothing has 
been taken for many hours. Moreover, small masses of mucus, which oc- 
casionally assume a snail-like spiral form, and saliva which is recognized by 
the presence of large flat epithelial cells and the so-called salivary corpuscles 
are quite frequent. A few bacilli and yeast cells are almost always observed. 

As such elements as the ones above mentioned are present in all gastric 
juice, we must not attach undue importance to the presence of small amounts 
of such material in the contents obtained after a test meal. After the Ewald 
meal one rarely finds anything beyond the presence of numerous starch 
granules and more or less mucoid material, along with bacteria of the various 
types which flourish particularly in the buccal and gastric cavities.^ 

In cases associated with diminished motility of the stomach we niay find 
remains of food which has been introduced many hours previously. In such 
specimens we observe numerous fat globules or fatty acid crystals, many vege- 
table fibers and cells and a few red blood-cells which have come from slight 
abrasion of the mucous membrane of the pharynx by the stomach-tube. 
These red cells usually are much altered in appearance by the hydrochloric 
acid and do not show their ordinary hemoglobin color, but take on a more 
brownish tint, which is due to the presence of hematin. 

Boas-Oppler Bacillus. 

This organism is found quite commonly in patients suffering with carci- 
noma of the stomach, and is almost always absent in nonmalignant disease. 
It is found more frequently in the gastric contents at a time when lactic acid 
is present in large amounts, so that in the incipient stages of carcinoma these 
organisms may be absent. These bacilh are very long (3 to 10 microns), 
I micron broad, and are frequently joined end to end forming very long chains. 
They are readily stained with the usual aniline dyes and by Gram's method 
and, on treatment with iodin, take on a brown color which distinguishes them 
from the large mouth bacillus (leptothrix buccalis), which stains blue with 
iodin. This organism appears to be identical with the bacillus bulgaricus, 
one of the lactic acid group, and is not an organism sui generis} It is not 
absolutely pathognomonic of carcinoma of the stomach, although Smithies^ 

^ See Lyon, Am. Jour. Med. Sc, 1915, CL, 402. 

^Galt and lies, Jour. Path, and Bacteriol., 1914, XIX, 239; Heinemann and Ecker, 
Jour. Bacteriol., 1916, I, 435. 

2 Jour. Am. Med. Assn., 1913, LXI, 1793. 



GASTRIC CONTENTS 63 

demonstrates its presence in 93.8 per cent, of 566 cases of pathologically 
verified carcinoma. Owing to the absence of hydrochloric acid in such cases, 
this organism develops and produces the lactic acid found. 

Sarcinae. 

Occasionally in normal gastric juice and especially in cases of dilatation 
with marked fermentation one finds the so-called sarcincB ventriculi which 
are cocci arranged in squares or tetrahedra which resemble, very much, 
cotton bales. These organisms have no pathologic significance, but are 
indicative of stagnation of gastric contents. Along with these sarcinae 
one may find large numbers of yeast cells. ^ 




Fig. 16. — Boas-Oppler bacilli. {Hemmeter.) 

Protozoa. 

These unicellular parasites have been occasionally found in the gastric 
contents. Flagellates, amebas, and monads seem to be more frequent than 
the other types of protozoa.^ They seem to be more commonly found in 
cases of carcinoma of the stomach. 
Fragments of Tissue. 

Frequently small shreds of mucous membrane are found in the expressed 
gastric contents. One finds these in cases of chronic gastritis, ulcer, hyper- 
chlorhydria, and especially in cancer. These tissue fragments should be 
studied carefully under the microscope, as not infrequently a diagnosis of 
cancer is possible from such examination. 
Crystals. 

Various types of crystal are occasionally noted in the gastric contents, 
among which may be mentioned bile acids, cholesterin, fatty acids, leucin, 
tyrosin, and calcium oxalate. If the reaction of the juice is alkaline triple 
phosphate crystals may appear. 

V. Chemical Examination 

The chemical examination of the gastric juice is the most important of all 

laboratory methods in the diagnosis of various pathologic gastric conditions. 

^ See Anderson, Jour. Infect. Dis., 191 7, XXI, 341. 
2 See Smithies, Am. Jour. Med. Sc, 191 2, CXLIV, 82. 



64 DIAGNOSTIC METHODS 

As previously stated, the acidity of gastric juice is referable to the presence of 
free and combined acids. The free acidity is traceable largely to hydrochloric 
acid, although organic acids, such as lactic, acetic, and butyric, may increase 
the free acidity under abnormal conditions. Besides this free acidity, we have 
hydrochloric acid which is bound chemically to the protein substances and 
does not react with tests for free acidity. There are also present in the gastric 
juice acid salts, especially the sodium dihydrogen phosphate (NaH2P04). 

Besides these factors which have to do with the reaction of the gastric 
juice, we find certain ferments which act only in the presence of the free hy- 
drochloric acid.^ The first of these, pepsin, has the power of acting upon al- 
bumin in an acid medium and converting it, through various stages, into lower 
splitting products of albumin. This peptic digestion will be discussed in 
detail later. A second ferment, known as rennin, lab, or chymosin, has the 
power of curdling milk by coagulating the casein. A third ferment, lipase,^ 
acts upon fat, especially when this is present in a finely divided form. This 
lipolytic action is not ordinarily great, but should nevertheless be remembered. 
The experiments of Sahli show that this action is negligible during the period 
covered by the administration of his test meal. 

Although many statements have been made to the contrary, the gastric 
juice, through the agency of the hydrogen ions of its free hydrochloric acid, 
acts upon certain polysaccharides, especially cane sugar, hydrolyzing them 
into the simpler monosaccharides. Careful determinations indicate that the 
speed of inversion is about the same as that of an equal strength of hydrochloric 
acid, so that we do not need to assume any ferment action. 

Besides these substances gastric juice contains a small amount of albumin, 
carbohydrates, and various inorganic salts. ^ 
(i) Total Acidity. 

As previously stated, the total acidity of the gastric juice is referable to the 

presence of free and combined hydrochloric acid, organic acids, -and acid salts. 

This factor may be readily determined by titrating lo c.c. of gastric juice 

with tenth-normal sodium hydrate, using phenolphthalein as an indicator. 

This indicator is colorless in the presence of acid and becomes red at the point 

of neutralization, being used as a i per cent, alcoholic solution. On adding a 

few drops of this solution to the unfiltered gastric contents, a white cloud will 

be observed due to the precipitation of the reagent by the water of the gastric 

juice. The titration is carried to the point at which the addition of sodium 

hydrate produces a definite pink color which remains permanent and does not 

deepen on the addition of further alkali. If sodium chlorid be added to the 

point of saturation of the gastric contents, the end point becomes somewhat 

sharper owing to the diminished dissociation which the disodium hydrogen 

phosphate undergoes into sodium dihydrogen phosphate in the presence of 

increased sodium ions. This precaution is rarely taken, however, as the 

clinical result is never so accurately determined as is the scientific factor. 

^ See Ewald, Deutsch. Arch. f. klin. Med., 1912, CVI, 498. 
- See Hull and Keeton, Jour. Biol. Chem., 191 7, XXXII, 127. 

3 See Huber, Am. Jour. Physiol., 191 7, XLII, 404, as to origin of the gastric ammonium 
salts. 



GASTRIC CONTENTS 65 

The total acidity varies between rather wide limits. Normally it ranges 
from 75 to 100°, being made up of approximately 50° of free hydrochloric acid, 
25° of combined hydrochloric acid, and 25° of organic acids and acid salts. 
The chief variation under normal conditions is an increase in the combined 
hydrochloric acid and a decrease in the organic acids and acid salts. 

In pathologic conditions we may find the total acidity high with very 
little free hydrochloric acid, or we may find the total acidity low, with a 
normal amount of hydrochloric acid present. 

Sahli has stated the variations in the acid factors of the stomach contents 
as follows: If the total acidity is high and the hydrochloric acid is normal, 
the high acidity can be due only to a deficient motility and absorption and 
hence we find an increase in the organic acids. Such a gastric juice may show 
lactic acid, but will more probably give the tests for the other organic acids. 
A low total acidity with an excess of hydrochloric acid shows that the 
motility and absorptive powers of the stomach are good. If the total acidity 
be moderate and free acid small in amount, a poor motility may be assumed. 
Generally speaking, when much lactic acid is present we find low HCl and 
only combined HCl; that is, diminished secretion and diminished motility. 
No lactic acid is found when the HCl is normal or increased. 

(2) Free Hydrochloric Acid. 

A number of tests have been devised for the detection of free hydrochloric 
acid in the stomach and its differentiation from lactic and acetic acids. It 
should be remembered that the tests outhned below are not specific tests 
for hydrochloric acid, but are common to all mineral acids. Many of these 
tests react also with the organic acids, providing they are present in sufficient 
concentration. The efficiency of any acid is due merely to the ionic decom- 
position which it suffers when in solution; in other words, is due to the 
presence of free hydrogen ions. Hydrochloric acid appears to be more effi- 
cient in the digestive processes than do the organic acids, owing simply to its 
greater degree of dissociation. The tests commonly employed in clinical 
work are based upon the reaction which certain coloring matters undergo 
when treated with free hydrochloric acid. These tests can be, therefore, 
only approximate and must be used with discretion in scientific work, al- 
though in clinical work they are near enough for all purposes. Were we able 
to express completely the stomach contents and thus to obtain material 
which would give us absolute data, we would then require better clinical 
methods. What should be measured in testing stomach contents for free 
acidity is the number of hydrogen ions which the gastric juice contains, since 
this is the important factor in the efficiency of the juice. ^ This may be deter- 
mined directly by measuring the speed of any catalytic reaction due to hydro- 
gen ions, the most convenient one being the rate of inversion of cane sugar. 

It is wise to have some quick method by which one may determine the 
presence of a free acid in the gastric juice. This may be done with litmus 

^ See Michaelis, Die Wasserstoffionen Konzentration, 1914, Springer, Berlin; Menten 
(Jour. Biol. Chem., 1915, XXII, 341) shows that the Ph of undiluted normal gastric juice, 
under varying conditions, ranged from 0.92 to 1.58. 

5 



66 DIAGNOSTIC METHODS 

which will, however, not show the presence of free hydrochloric acid to the ex- 
clusion of other acids or acid salts. To determine whether the acid reaction 
is due to free acid it is customary to employ the Congo-red paper. This con- 
sists simply of filter-paper which has been saturated with an alcoholic solution 
of Congo-red and dried. On treating gastric juice with such paper we obtain 
a blue color in the presence of free acid.^ This paper reacts with a blue color 
to any free acid so that one should never assume the presence of free hydro- 
chloric acid when he obtains a blue coloration. Although many writers state 
that a blue coloration is never given by free organic acids, the writer has seen 
too many cases in which distinct action was referable either to free lactic or 
free acetic acids to agree with this statement. The tests which are applicable 
to the detection of free mineral acids in general may be used as indicative of 
free hydrochloric acid, as this is the only mineral acid which one would 
ordinarily find in the gastric contents. 

Qualitative Tests. 

(a) Topfer's Test. 

The test is based upon the coloration which a 0.5 per cent, alcoholic solu- 
tion of dimethyl-amido-azobenzol takes when treated with gastric juice con- 
taining free hydrochloric acid. A few c.c. of unfiltered gastric juice are 
placed in a dish and one or two drops of the above solution added. In the 
presence of free mineral acids a carmin red color is obtained. This reagent 
is a very delicate one and does not react to organic acids unless they are 
present in amounts exceeding 0.5 per cent. The coloration with free hydro- 
chloric acid varies in intensity with the amount of acid present, and may range 
from a deep orange to an intense carmin. According to Simon, lactic acid 
does not give the t3rpical red color with this reagent, especially if albumoses 
are present, unless it be in a concentration of at least i per cent. This 
reagent will detect the presence of 0.02 parts of hydrochloric acid per thousand . 

(b) Giinzburg's Test. 

The reagent employed in this test consists of 2 grams of phloroglucin and 
I gram of vanillin dissolved in 30 c.c. of absolute alcohol. This yellowish solu- 
tion should be kept in dark bottles, as it gradually changes to a dark red and 
then to brown when exposed to the Hght. Boas claims that the reagent be- 
comes more delicate and stable if one dissolves the phloroglucin and vanillin 
in 100 c.c. of 80 per cent, alcohol. 

Two or three drops of this solution are added to an equal amount of the 
gastric juice contained in a porcelain dish and the mixture evaporated over a 
water-bath. In the presence of free mineral acid a rose-red color is developed, 
varying in intensity with the amount of acid present. This mixture must not 

^Holmgren (Deutsch. med. Wchnschr., 1911, XXXVII, 247) has devised a quantitative, 
as well as qualitative, test for free HCl using the capillarity of such Congo-red paper as a 
basis of estimating the quantity of HCl, the discolored zone varying in width with the degree 
of acidity. This method of "capillary analysis" is, however, not as constant and reliable 
for quantitative purposes as are the titration methods, as Mattisson (Arch, f . Verdauungskr., 
1913, XIX, 79 and 226) and Schmidt (Biochem. Jour., 1913, VII, 231) have shown.^ Aberg 
(Hygiea, LXXVI, 105) advises the use of tincture of cochineal or lacmoid as an indicator in 
this test. See, also, Sochanski, Arch. f. Verdauungskr., 1914, XX, 317. 



GASTRIC CONTENTS 67 

be boiled or heated too rapidly as the resulting color will then be brown or 
brownish-red and may mislead one into beheving that no free hydrochloric 
acid is present. 

This test does not react to organic acids or to acid salts, nor is it interfered 
with by the presence of products of food digestion. It may, therefore, be used 
with the unfiltered gastric juice. This test reacts with the rose-red color in 
the presence of 0.05 parts of HCl per thousand. - 

(c) Boas* Test. 

This reagent consists of 5 grams of resorcin and 3 grams of cane sugar 
dissolved in 100 grams of 95 per cent, alcohol. It has the same delicacy as 
Giinzburg's test and is more stable. The test is applied in the same way as 
the preceding, taking particular care to use a low flame in evaporating, and 
gives a rose-red or vermilion color in the presence of mineral acids. This color 
gradually fades on cooling and is not given by organic acids or acid salts. 

(d) Tropeolin Test. 

The reagent for this test is a saturated alcoholic solution of tropeolin 00. 
This test is applied in the same way as the preceding and gives a lilac-blue 
color in the presence of free acid.^ This test is not as delicate as the preced- 
ing, reacting only in the presence of 0.3 parts of free hydrochloric acid per 
thousand, and has the objection that it strikes the blue color much more easily 
with the organic acids. 

Other tests have been advocated for the quahtative detection of free 
hydrochloric acid, but they are not as delicate as the above and have nothing 
to justify their existence.^ Of the tests given, the Giinzburg test seems to be 
the most rehable, although the Topfer's test is clinically sufficient and has the 
advantage of being much less expensive and more convenient than is the for- 
mer reagent. 

Quantitative Methods. 

The quantitative estimation of free hydrochloric acid is of great import- 
ance in the study of all pathologic conditions of the stomach. Any determi- 
nation made with our present methods must have reference to the fact that 
[.the acidity of the gastric juice is due almost entirely to the free hydrochloric 
'acid. While this is not absolutely true, yet the organic acids are rarely pre- 
(sent in sufficient amounts to react with the indicators in conditions in which 
hydrochloric acid is normal or increased in amount. However, in the condi- 
tions associated with diminished amount of hydrochloric acid one must be on 
his guard in the interpretation either of the qualitative or quantitative tests 
[for free hydrochloric acid. The writer has seen several cases in which all of 
the indicators, with the exception of Giinzburg's reagent, showed positive re- 
sults for hydrochloric acid, the cases being those in which no free hydrochloric 
acid was actually present. Bearing this in mind we may determine the 
acidity, referable to free hydrochloric acid, by the titration of a known amount 

^See Christiansen, Biochem. Ztschr., 1912, XL VI, 24, 50 and 71. 

2 Fittipaldi (Gazz. d. osp., 1913, XXXIV,.289) uses this reagent for quantitative purposes. 

^See, however, Cipallina, Riforma Med., 1913, XXIX, 505. 



68 ■ DIAGNOSTIC METHODS 

of the unfiltered gastric juice with tenth-normal sodium hydrate solution, using 
as indicators the solutions mentioned under the head of qualitative tests. ^ 

Mintz Method. 
As the Giinzburg reagent is the most delicate and reliable of all the tests 
for free hydrochloric acid, it is wise to use this reagent as an indicator. The 
test as usually followed by the writer is to add 20 to 30 drops of the Giinzburg 
reagent to 10 c.c. of the gastric juice. On adding the sodium hydrate solution 
no color change will be visible, as the reaction takes place only when the solu- 
tion is warmed. This warming cannot be done directly as the evaporation 
would necessarily have to proceed to the point at which loss of hydrochloric 
acid might occur. Following the recommendation of Sahli, the rod with 
which the solutions are stirred is warmed before being used. As the neutrali- 
zation point is reached the red color, originally noted on the sides of the warm 
rod, disappears. 

Topfer's Method. 
This method is the simplest, is the most generally used, and at the same 
time is one of the most delicate of all the chnical quantitative methods for 
free hydrochloric acid. It consists in the use of dimethyl-amido-azobenzol as 
an indicator, the titration of the unfiltered gastric juice being done with tenth- 
normal sodium hydrate solution. Although this reagent does give, under 
some conditions, a red color in the absence of free hydrochloric acid when 
the organic acids are largely increased, yet such conditions are so rarely found 
in clinical work that the result of test-tube experiments cannot be apphed to 
cHnical cases. The experiments of Simon show that lactic acid must be 
present to the extent of i per cent, before any cherry-red color is obtained, 
providing albumoses are present. As these latter substances are always 
found in the gastric juice after intake of protein material, one can readily see 
that lactic acid need not be considered. We find, however, conditions asso- 
ciated with fermentative processes in the stomach in which acetic and butyric 
acids are present in fairly large amounts. These acids will give a red color 
with the indicator, but should not mislead as their strong odor in such con- 
centrations permits of easy recognition. 

In the titration the sodium hydrate solution is added from a buret to the 
unfiltered gastric juice to which one or two drops of indicator are added for 
every 10 c.c. of juice. In the presence of free hydrochloric acid this indicator 
strikes a distinct cherry-red tone and thus enables the worker to decide at 
once as to the presence or absence of the acid. No evaporation is necessary, 
hence the test has the advantage of simplicity and does not occasion any loss 
of substance. As the sodium hydrate solution is added the reddish tint of 
the mixture changes to a distinct yellow. The titration must be carried to 
the point at which every trace of red disappears and the color becomes a pure 
yellow. This reaction requires considerable experience, hence the writer 
would advise the student to make his titration with a known solution of hy- 
drochloric acid so that he may become familiar with the end point. 

Although the writer formerly used the filtered contents in his estimations, 
^ See Knapp, New York Med. Jour., 1913, XCVII, 437. 



GASTRIC CONTENTS 69 

he finds that filtration causes distinct changes in the composition of the fluid, 
owing to adsorption of free hydrochloric acid. He advises, therefore, the use 
of the thoroughly mixed unfiltered contents, unless there is a very large 
amount of mucus present. Under this latter condition one may use the 
filtered contents. If one wishes to clarify the fluid for the determination of 
pepsin, it is wise to centrifuge the specimen instead of filtering.^ 

Amount of Free Hydrochloric Acid. 

Although the general idea prevails that the normal human gastric juice 
contains free hydrochloric acid to the extent of 0.2 to 0.3 per cent., recent 
studies, under properly controlled conditions, on both lower animals and man, 
indicate that this amount reaches a constant figure more nearly that of 0.5 per 
cent, in cases in which the freshly secreted normal gastric juice is examined. 
Carlson^ calls attention to the fact that normal human gastric juice secreted at 
a fairly high rate is equal in total acidity to the acidity reported by clinicians 
for so-called hyperacidity, and states that "there is no evidence that the gas- 
tric glands under any pathologic conditions are able to or do secrete a juice of 
higher than normal acidity." Although it is impossible to deny the existence 
of cases of hyperacidity, these experiments and conclusions are, to a certain 
extent, in conformity with cUnical observations, as we unquestionably ob- 
, serve many cases of high acidity with no symptoms of this condition and, on 
the other hand, symptoms of hyperacidity in cases of real hypoacidity. 
Hence, we are forced to the conclusion that the same amount of acid may indi- 
cate a hyperacidity in one case and not in another. The fallacy of relying on 
percentage relations instead of relating the symptoms of hyperacidity to a 
hypersensitiveness of the gastric mucosa toward certain degrees of hydro- 
chloric acid is, therefore, evident. 

This normal acidity does not remain in the digesting stomach at this 
higher level for a great length of time, being relatively soon lowered to a level 
of optimum acidity of about 0.2 to 0.3 per cent., according to Boldyreff,^ by 
neutralization by the mucus secreted by the gastric glands, by the combina- 
tion of the acid with the protein of the food and, especially, by reflux of alka- 
line juices from the duodenum into the stomach. At the height of digestion 
we find, therefore, that the acidity of normal gastric juice may range from 0.2 
to 0.5 per cent., this optimum varying in different subjects and with the type 
of food in the stomach. Under ordinary conditions this excess of free hydro- 
chloric acid should be evident in 45 to 60 minutes after an Ewald breakfast, 
but this time varies with the individual case. It is evident, therefore, that a 
diagnosis of hyperacidity or hypoacidity cannot be absolutely made by refer- 
ence simply to the chemical analysis of a gastric contents.* 

Certain clinical conditions lead to an excretion of gastric juice which is 
normal, increased, or diminished in amount of free hydrochloric acid. We 

^ See Christiansen, Biochem. Ztschr., 1912, XL VI, 82; Zunz, Handb. der Biochem. 
Arbeitsmeth., 1914., VIII, 44; Seidl, Arch. f. Verdauungskr., 1915, XXI, 196. 

2 Am. Jour. Physiol., 1915, XXXVIII, 248; also, Menten, Jour. Biol. Chem., 1915, XXII, 
341. 

^ Quart. Jour. Exper. Physiol., 1914, VIII, i. 

^ See Rehfuss and Hawk, Jour. Am. Med. Assn., 1914, LXIII, 2088; Yagiie, Arch. Esp. 
de Enf. del ap. Dig., 1919, II, 528. 



70 DIAGNOSTIC METHODS 

must, therefore, have a method of interpretation of the free HCl acidity of 
the stomach. It is customary to report the results of titration of a given 
specimen of gastric juice in one of two ways: 

(i) We may represent the acidity referable to free hydrochloric acid by 
the number of c.c. of tenth-normal sodium hydrate necessary to neutralize loo 
c.c. of unfiltered gastric juice, using dimethyl-amido-azobenzol as an indi- 
cator. This is called degree or percentage of acidity. Thus, if 5 c.c. of tenth- 
normal sodium hydrate were used to neutrahze 10 c.c. of unfiltered gastric 
juice, the degree or percentage of acidity would be, obviously, 50. 

(2) We may report the actual amount of free hydrochloric acid present. 
This is the more scientific way, as we have a much better means of comparison 
with the normal standards. One c.c. of tenth-normal sodium hydrate neu- 
tralizes 0.00365 gram of free HCl. If, now, we multiply this factor by the 
number of c.c. necessary to neutralize 100 c.c. of unfiltered gastric juice, we 
obtain a figure representing the absolute amount of free HCl in the gastric 
content. Thus, if the gastric juice showed an acidity of 50 degrees, we would 
have 0.1825 (50 X 0.00365) gram of HCl. The usual text-book statement is 
that the free hydrochloric acid is normally about 40 degrees, but the writer is 
accustomed to consider an acidity of 50 to 55° as much more nearly normal 
than one of 40. 

Euchlorhydria. 
This is a condition in which the amount of free hydrochloric acid is be- 
tween 0.2 and 0.3 per cent. In speaking of percentage one must not confuse 
the two types of percentage reckoning. Thus, 10 per cent, or 10° of HCl rep- 
resents only 0.0365 gram or true per cent, of hydrochloric acid. The lower 
figure of 0.2 per cent, is given for euchlorhydria owing to the fact that normal 
variations may permit .of this low point, although the higher figure of 0.3 per 
cent, is the more usual. When this euchlorhydria exists in the presence of 
clinical symptoms pointing to gastric disturbance, we usually have to do with 
a neurosis. Gastritis may be absolutely ruled out, a carcinoma excluded ex- 
cept when the new growth has taken place upon an old ulcer and an ulcer prac- 
tically always ruled out, although this latter may show an euchlorhydria. 
This condition may be associated with a certain amount of atony along with 
more or less marked dilatation. 

Hypochlorhydria. 
This is a condition associated with excretion of the gastric juice showing 
o. I or a lower per cent, of hydrochloric acid. It is found especially in sub-acute 
or chronic gastritis, in incipient carcinoma, in fevers, severe anemias, many 
mental diseases, passive congestion due to valvular heart lesions, many cases 
of chronic nephritis, and dilatation of the stomach. Some rare cases of ulcer 
of the stomach show a low degree of free hydrochloric acid, but this is not usual. 

Anachlorhydria. 
This is a condition characterized by the excretion of the gastric juice show- 
ing the complete absence of free hydrochloric acid. This condition has been 
supposed to be pathognomonic of gastric carcinoma. There are, however, 
many cases of cancer which show either a hypo- or a hyperchlorhydria, and 



GASTRIC CONTENTS 7 1 

there are, also, man}' other conditions which show an anachlorhydria. Thus 
we find this condition in a large majority of cases of advanced chronic gastri- 
tis, in the severe anemias, especially of the pernicious type, in neurasthenia 
and hysteria, in many severe febrile diseases, and in atrophic gastritis.^ 
Hyperchlorhydria. 

This condition exists when we have the excretion of a gastric juice showing 
more than 0.2 per cent, hydrochloric acid. This figure may run anywhere 
from 0.2 too.9 per cent.^ It is a very common occurrence in nervous individ- 
uals, in ulcer of the stomach, some cases of chlorosis, in some chronic cach- 
exias, in the early stages of chronic gastritis, in carcinoma which is grafted on 
to an old ulcer, in continuous hypersecretion, in chronic passive congestion of 
the stomach, and in cases of migraine.^ 

It will thus be seen that the hydrochloric acid of the gastric juice varies 
under clinical conditions to quite an extent. While an increased amount of 
hydrochloric acid is usually present in ulcer of the stomach, we must not 
necessarily make our diagnosis on this point alone. Likewise in carcinoma of 
the stomach we should not exclude this condition if the examination of the 
stomach contents does not show a lessened amount of hydrochloric acid or 
even a total absence, as many cases of carcinoma may show all varieties of 
chlorhydria. 
(3) Combined Hydrochloric Acid. 

As the hydrochloric acid first secreted by the gastric glands combines with 
the protein of the food material, it is necessary to have some method by which 
we may determine just how much of this material has been formed in the 
stomach. The physiologically active hydrochloric acid consists of both the 
free and combined acid so that we may have only a slight amount of the free 
acid, but a relatively large amount of the combined. Not infrequently we 
find cases which show no free hydrochloric acid, but quite a percentage of the 
combined acid, indicating that a certain amount of acid has been secreted by 
the gastric juice. This combined hydrochloric acid has, therefore, a certain 
clinical importance and should be investigated in every case.^ No direct 
methods are known for its determination so that we must resort to indirect 
methods. 
Method of Martius and Liittke. 

This method, as modified by Reissner, is much more applicable to scien- 
tific work than to clinical investigations, as it is too complicated and time-con- 
suming for the ordinary practitioner. It is based upon the facts that the free 
hydrochloric acid as well as the acid combined with protein material escape 
upon incineration of the gastric juice, while the inorganic chlorin in combina- 

^Ohly (Deutsch. med. Wchnschr., 1913, XXXIX, 1402; Arch f. Verdauungskr., 1915, 
XXI, 128) calls attention to the fact that 70 to 80 per cent, of all cases of progressive chole- 
cystitis^ show an anacidity or actual achylia, although a shght hyperacidity may obtain in 
the incipient stages. This is of importance in differentiating a pyloric or duodenal ulcer 
from cholehthiasis. See, also, Hernando, Arch, des Mai. de I'App. digestif, 19 14, VIII, 
274; Griinfelder, Ztschr. f. exp. Path. u. Ther., 1914, XVI, 141. 

^ See page 09. 

^Surmont and Dehon (Arch, des Mai. de 1 App. digestif, 1914, VIII, 246) believe that 
defective ehmination of salt by the kidneys is often compensated for by hyperchlorhydria. 

" See Grund, Deutsch. Arch.f. klin. Med., 1913, CIX, 560. 



72 DIAGNOSTIC METHODS 

tion with inorganic bases remains in the ash. If the total amount of chlorin 
present in the filtered gastric juice be determined (a), and then the amount of 
chlorin in the ash (b) investigated, subtraction of the latter (b) from the for- 
mer portion (a) will give the amount of chlorin referable to free and combined 
hydrochloric acid except a small loss referable to volatilization of ammonium 
chlorid. If the gastric juice be neutrahzed with sodium hydrate before it is 
incinerated and the chlorin in this ash determined, the amount of this chlorin 
(a') subtracted from (a) represents the ammonium chlorid volatiHzed. 
Hence a' — b equals the free and combined HCl. By now determining the 
amount of free hydrochloric acid according to Topfer's method we may at 
once calculate the combined hydrochloric acid by subtracting this result from 
the amount of free and combined hydrochloric acid previously obtained. 
The method of determining the chlorin will be fully discussed in the section 
on urine to which the reader is referred. 
Method of Topfer. 

This method embraces three separate determinations. In the first place 
the total acidity of the gastric juice is determined by titration of lo c.c. of 
unfiltered gastric juice with tenth-normal sodium hydrate, using phenolphtha- 
lein as an indicator. This result is termed a. This indicator has the advan- 
tage of reacting toward anything of an acid nature, and will give us, therefore, 
the different factors which go to make up the total acidity of the gastric juice, 
namely, the free and combined acids, the organic acids, and the acid salts. 

Having determined this factor (a), a second portion of lo c.c. of gastric 
juice is titrated with tenth-normal sodium hydrate solution using a i per cent, 
aqueous solution of alizarin (aHzatin monosulphonate of sodium) as an indi- 
cator. Two or three drops of this indicator are added to lo c.c. of unfiltered 
gastric juice when the mixture becomes distinctly yellow. The titration is 
carried to the point of production of a pure violet color which does not deepen 
on the further addition of alkali. As this reaction demands the recognition of 
a change from yellow through a faint violet to a deep violet color the worker 
must have considerable practice before he is able accurately to determine the 
end point. The result is termed h. No trace of red should be present, a pure 
violet color being the true reacting point. This color may be observed by 
treating a few drops of alizarin solution with a i per cent, solution of sodium 
carbonate. Alizarin reacts with free acid, both mineral and organic, and with 
acid salts, but not with organically bound HCl. If, therefore, we subtract the 
figure obtained when alizarin is used as an indicator {h) from that obtained 
with phenolphthalein {a) the result will be the combined hydrochloric acid 
{a - h). 

If now we add this combined hydrochloric acid to the free hydrochloric 
acid, which has been obtained by titration of the gastric juice using dimethyl- 
amido-azo-benzol as an indicator, (c), we obtain the total physiologically ac- 
tive hydrochloric acid (c -j- (a — h)). The difference between the total acid- 
ity and this factor gives us the amount of organic acid and acid salts present. 
{a — {c -\- {a — h)). If but a small amount of gastric juice be available for 
chemical examination, recourse may be had to a modification suggested by 



GASTRIC CONTENTS 73 

Einhorn. This is a double titration of the same portion of juice. A few c.c. 
(5) of unfiltered gastric juice are treated with a few drops of dimethyl-amido- 
azobenzol and the solution titrated for free HCl with sodium hydrate. When 
the point of neutralization of the free acid is reached a few drops of phenol- 
phthalein solution are added and the titration continued to the point of neu- 
tralization of total acidity. These indicators do not interfere at all with one 
another as their reacting points are usually widely different. The writer has 
found this method reliable and convenient. 

Hydrochloric Acid Deficit. 

In those cases in which the gastric contents show no free hydrochloric acid, 
it is customary to determine the HCl deficit. By this is meant the amount of 
hydrochloric acid which must be added to the gastric contents before it shows 
a reaction for free acid. This amount will naturally depend on the amount 
of combined HCl already present, the amount of protein in the gastric con- 
tents and the amount of alkali secreted. Sahli suggests the expression "satura- 
tion deficit" for this figure. Ten c.c. of unfiltered gastric juice are titrated 
with tenth-normal hydrochloric acid, using dimethyl-amido-azobenzol as an 
indicator and titrating to the point of production of the red color. The result 
is expressed in terms of degrees as under the representation of the free hydro- 
chloric acid. This factor enables one to follow the course of the disease, 
showing how little hydrochloric acid is excreted for combination with the 
proteins of the food. 

(4) Organic Acids. 

The organic acids, outside of the lactic acid, have very little clinical signifi- 
cance. The food practically always contains a certain amount of fatty acids 
which appear in the stomach contents and contribute to the total acidity. In 
the normal digestion of the carbohydrates, lactic acid is practically always 
formed, so that excess of this acid would indicate excessive fermentative proc- 
esses in the stomach, due to a combination of diminished amount of hydro- 
chloric acid along with a lessened motility of the stomach. Other acids, such 
as butyric and acetic acids, are formed in this same process of carbohydrate 
fermentation, so that the organic acids may represent a large portion of the 
total acidity. Besides this, bacterial decomposition, in the absence of hy- 
drochloric acid, plays a role in the production of these fatty acids. The fat- 
sphtting ferment, lipase, may produce these organic acids in fairly large 
amounts. 

Total Organic Acid. 

It is sometimes of importance to know just how much organic acid is pres- 
ent in the stomach contents. This may be done directly by the Hehner-Maly 
method, which is based upon the fact that, if a mixture of organic and inor- 
ganic acids be neutralized and then incinerated, the organic acids will be con- 
verted into carbonates while the inorganic acids remain as neutral salts. If 
the alkalinity of these carbonates be then determined and this factor sub- 
tracted from the total acidity we obtain directly the mineral acids. This is 
possible owing to the fact that the degree of alkalinity of the carbonates is 



74 DIAGNOSTIC METHODS 

equal, in terms of tenth-normal solutions, to the acidity referable to the 
organic acids. The technic is as follows: The total acidity of lo c.c. of 
gastric juice is determined by titration with* tenth-normal sodium hydrate 
solution as previously described. The neutralized solution is evaporated to 
dryness in a platinum dish and is then incinerated. The ash is dissolved in 
distilled water and the alkaline solution titrated with tenth-normal oxalic 
acid solution. As i c.c. of the tenth-normal oxalic acid solution is equivalent 
to I c.c. of tenth-normal sodium hydrate solution, we subtract the factor 
obtained in the latter titration from that of the former and obtain directly 
the degree of acidity due to mineral acids. The number of c.c. of tenth- 
normal oxalic acid used represents directly the total organic acids present. 

In this method the acid salts are included in the factor referable to mineral 
acids, so that we may subtract from this factor the degree of acidity, attribut- 
able to free hydrochloric acid, and obtain the amount of acid salts present. 
In some cases fatty acids are present which are not soluble in water and conse- 
quently are not neutralized by the addition of the sodium hydrate solution. 
These acids may be extracted from the neutralized solution with ether and 
may then be neutralized and added to the neutral aqueous solution. The 
mixture is now evaporated as before and incinerated. This estimation of the 
higher fatty acids requires the use of the unfiltered gastric contents. However 
such acids do not play a large clinical role and may be ordinarily omitted. 

(a) Lactic Acid. 

The ordinary foods such as milk, bread, and meat contain a certain 
amount of lactic acid, so that any test for the presence of lactic acid can be 
of value only when the meal contains very little of such foods or when the 
portion taken in with the food has disappeared from the stomach. After 
the Boas meal there is always less lactic acid than after the Riegel meal, 
so that the former is much preferable when a special test is to be made for the 
presence of lactic acid. Boas has shown that under physiological conditions 
no appreciable amount of lactic acid is formed during the process of digestion. 
At the height of digestion practically no lactic acid is demonstrable in the 
stomach contents. This may be due to its absorption, on the one hand, or, 
on the other, to the fact that the hydrochloric acid interferes with the delicacy 
of the reactions. Pathologically, lactic acid is found in any condition associ- 
ated with stagnation of the gastric contents as a result of motor insufficiency, 
provided the amount of hydrochloric acid is below the normal amount. As 
this condition of affairs is found most frequently in cases of carcinoma of the 
stomach, an excess of lactic acid is very strongly suggestive of malignancy, 
although it must be remembered that such an excess may appear in cases of 
benign stenosis and gastric insufficiency.^ If the stomach be washed out the 
evening before giving a test meal, preferably the Boas meal, and lactic acid 
be found in appreciable amounts, carcinoma is the probable diagnosis. This 
finding of increased lactic acid and diminished hydrochloric acid is not always 
observed in every case of carcinoma of the stomach. In some cases periods 
of increased production of hydrochloric acid alternate with increased forma- 

1 See Rodella, Cor.-Bl. f. Schweiz. Aerzte, 1918, XL VIII, 1210; Ibid., 1919, XLIX, 1623. 



GASTRIC CONTENTS 75 

tion of lactic acid, and in some cases, especially those in which the carcinoma 
has developed upon the base of an old ulcer, no lactic acid may be present, 
but hydrochloric acid may be found in large amounts. 
Uffelmann's Test. 
This test is, perhaps, more commonly employed for the detection of lactic 
acid than is any other, but the writer prefers the Kelling test. Uffelmann's 
reagent consists of 20 c.c. of i per cent, carbolic acid solution, to which are 
added one drop of dilute ferric chlorid solution and sufficient water to form a 
transparent amethyst-blue solution. This solution is not permanent and 
must, therefore, be made fresh before each test. If a few drops of the filtered 
gastric juice be added to 5 c.c. of this reagent, the solution will be decolorized 
in the presence of lactic acid, taking on a beautiful canary-yellow or greenish- 
yellow tint. The mere decolorization of this solution is not sufficient for a 
positive test. A pure lemon yellow or canary color must be present before 
one may assume the presence of lactic acid. Even when this color appears 
one must eliminate such factors as the acid sodium phosphate, cane sugar, 
glucose, alcohol, and various oiiganic acids, such as tartaric, citric, or oxalic, 
before he can say that lactic acid is present. A considerable excess of hydro- 
chloric acid in the gastric juice may prevent the appearance of this color and 
likewise a yellowish tint of the stomach contents may obscure the result. 
Under such conditions it is necessary to extract the gastric contents with ether, 
which takes up the lactic acid. The ethereal solution is then evaporated, the 
residue taken up with distilled water, and the test applied to this solution.^ 

Kelling's Test. 

This test is in reality a modification of the previous one and consists in the 
addition of a few drops of filtered gastric juice to a very dilute solution of ferric 
chlorid. As used in the writer's laboratory, the method is as follows: To 
a test-tube full of water are added one or at most two drops of a 10 per cent, 
solution of ferric chlorid. The mixture is thoroughly shaken and divided into 
two portions, one of which serves as a control. On now adding a few drops 
of filtered gastric juice to one of these portions a distinct canary-yellow color 
will appear in the presence of lactic acid. The color of the two solutions 
should be compared so that any change in the one, to which gastric juice was 
added, may be observed. This test has the same objections as the Uffel- 
mann test, so that it is always wise to extract the gastric juice with ether. 
Strauss' Method. 

This method is, perhaps, the very best clinical method at our disposal, 
as it shows lactic acid when present in pathological amounts. It does not, 
however, give a quantitative result, nor does one seem necessary in the 
ordinary clinical work. Into a special separatory funnel (see cut) are intro- 
duced 5 c.c. of the gastric juice. The funnel is then filled to the 25 c.c. mark 
with alcohol-free ether and well shaken. The ethereal layer will take up the 
lactic acid from the gastric contents. After the fluids have settled the gastric 
juice and ether are allowed to run out to the mark 5 by opening the stop-cock, 

^ _i Gerard and Regnoult, C. R. Soc. biol., 1918, LXXXI, 388, call attention to the possi- 
bility of errors arising from the presence of lactic acid in the bread of the test meal. 



76 



DIAGNOSTIC METHODS 



after which distilled water is added to make up the 25 c.c. volume. Two drops 
of 10 per cent, ferric chlorid solution are then added with a medicine dropper 
and the mixture well shaken. The water will now extract the lactic acid 
from the ether. The aqueous layer is colored an intense greenish-yellow 
if more than o.i per cent, of lactic acid is present, while smaller amounts 
will show a shght greenish tinge. This test may be negative if the lactic 
acid present is completely combined with the proteins of the gastric juice. In 
such cases hydrochloric acid may be added to liberate this lactic acid before 
shaking out with ether. 

Other qualitative tests as well as several quantitative 
tests have been given for lactic acid. Quantitative deter- 
minations of lactic acid do not seem to be of any great 
clinical importance, as any marked reaction for this sub- 
stance is indicative of a pathologic condition whose extent 
may bear no relation whatever to the amount of lactic acid 
present. A general idea of the amount of lactic acid may 
be obtained by evaporating 10 c.c. of gastric juice, acidu- 
lated with a few drops of sulphuric acid, to the consistency 
of a syrup and then extracting this residue several times 
with acid-free and alcohol-free ether. The ether may be 
removed by evaporation and the residue taken up with 
water. This watery solution may now be titrated with 
tenth-normal sodium hydrate, each c.c. of alkali used 
representing 0.009 gram of lactic acid. 

The method of Boas, while very exact, is much too 
complicated and time-consuming for clinical work so that 
the writer will refer to other books for a descriptix)n of 
this test. The principle of the method is based upon the 
fact that when the lactic acid is heated with a strong oxi- 
dizing agent it is decomposed into acetic aldehyd and formic 
acid. If now the aldehyd be distilled off and trasnformed 
into iodoform by the addition of alkaline iodin solution^ 
this iodoform may be quantitatively determined. 

(b) Butyric Acid. 

This acid does not occur in the gastric contents, under physiological con- 
ditions, unless much milk or carbohydrate food has been introduced. Fliigge 
has shown that butyric acid may be derived from lactic acid and conse- 
quently may be present under the same conditions in which lactic acid is 
found. As butyric acid may be introduced from without and may have been 
formed in the mouth, one should be careful in drawing conclusions as to the 
clinical significance of butyric acid. 

If present in any large amount, butyric acid may be usually recognized 
by its distinct odor which is that of rancid butter. This test may not be 
sufficient for the recognition of butyric acid so that it is advisable to shake out 
the gastric juice with ether, evaporate, and take up with water as described 
under Lactic Acid. If a small pinch of powdered calcium chlorid be added to 




Fig. 17. — Strauss 
separatory funnel. 
(Hemmeter.) 



GASTRIC CONTENTS 77 

this watery solution and the mixture warmed, butyric acid will separate from 
the fluid in the form of small fat drops which float on the surface and have a 
characteristic odor of rancid butter. 

If a portion of the dried ethereal extract of the gastric juice be treated with 
a few drops of concentrated sulphuric acid and a little alcohol, the odor of ethyl- 
butyrate is perceptible on slight warming. This odor is that of pineapples 
and is very easily recognized. This test is known as the pineapple test. 

(c) Acetic Acid. 

It is not an infrequent occurrence to find acetic acid in rather large amounts 
in pathological conditions. The pathologic acetic acid is formed by the 
bacterial decomposition of the alcohol which is produced by the action of yeast 
upon carbohydrates. As yeast fungi are so frequently present in cases of 
dilatation of the stomach, associated with stagnation of its contents, acetic 
acid may be found under such conditions and constitute a portion of the 
total acidity. 

In testing for acetic acid the aqueous extract of the ethereal residue of 
the gastric juice is carefully neutralized with sodium carbonate solution. 
If a few drops of ferric chlorid solution be added to this neutralized solution 
a deep red color will appear if acetic acid be present. If this solution be 
boiled a reddish precipitate of basic ferric acetate is formed. This neutrali- 
zation of the aqueous solution is an essential point in this test, as the presence 
of free acid will prevent the appearance of any precipitate and the presence 
of free alkali will cause the formation of ferric hydroxid which will mislead, 
as the coloration is very much the same. The writer has seen several cases in 
which acetic acid was mistaken for lactic acid when the Kelling test was ap- 
plied. On adding gastric juice containing a large amount of acetic acid to the 
dilute ferric chlorid solution a change in color is observed, but in no case do 
you get the distinct canary-yellow color which can be possibly referable to 
acetic acid. 

(5) Gastric Ferments. 
(a) Pepsin. 

The enzyme pepsin is the most important of the ferments occurring in 
the gastric juice. As previously stated this ferment is excreted in the form 
of the zymogen (pepsinogen or propepsin) by the chief cells of the fundus 
glands. It becomes active, that is, converted into pepsin, by the free hydro- 
chloric acid of the gastric juice. This ferment acts only in acid media and is 
destroyed by the presence of minute traces of alkali.^ Its action is continuous, 
a small portion being capable of digesting large amounts of albumin, providing 
the products of this digestion are gradually removed. Should the products of 
ferment activity remain in the stomach an undue length of time, this ferment 
will cease to be active owing to the accumulation of the products of its own 

^^Hamburger (Arch. Int. Med., 1915, XVI, 356) confirms the fact that sodium chlorid 
inhibits the action of pepsin as formerly suggested by Schiitz and Levites. See Hamburger 
and Halpern, Arch. Int. Med., 1916, XVIII, 228; Ringer, Kolloid. Ztschr., 1916, XIX, 253; 
Biedermann, Ferment-forsch., 191 7, II, i; Davis and Merker, Jour. Am. Chem. Soc, 1919, 
XLI, 221. See, also, Edie (Biochem. Jour, 1914, VIII, 193) for a discussion of the recip- 
rocal action of pepsin and trypsin. 



yS DIAGNOSTIC METHODS 

activity. Pepsin acts in the presence of many other acids, but the concen- 
tration of these acids must be higher than in the case of hydrochloric acid. 
Thus, a 0.2 to a 0.4 per cent, hydrochloric acid gives the best results with 
pepsin, while a i to a 1.018 per cent, lactic acid is necessary to bring about 
good results. 

Very little data exists as to the amount of pepsin or of its zymogen so that 
we are forced to draw our conclusion regarding a normal or abnormal amount 
of this ferment from the rate at which known amounts of albuminous material 
are digested. Pepsin acts only upon protein substances, giving rise to a 
series of decomposition products which will be discussed later. Normally 25 
c.c. of gastric juice will dissolve (digest) 0.05 to 0.06 gram of serum albumin 
in one hour, the same amount of coagulated egg-albumin in three hours, and 
a similar amount of fibrin in one. and one-half hours. 

A diminution in the amount of pepsin must be referable to a direct disease 
of the secreting gland, as general abnormalities do not affect this function 
as much as they do the production of free hydrochloric acid.^ Pepsin is 
usually present when the free hydrochloric acid is either increased or 
diminished, but in cases of carcinoma, atrophic gastritis, and in occasional 
cases of pernicious anemia we may find no pepsin and no hydrochloric acid. 
Such a condition is known as achylia gastrica and occurs sometimes as a direct 
pathologic condition without a known etiology. It frequently happens that 
pepsin is present when no hydrochloric acid is found. 

Qualitative Tests. ' 

The digestive power of the filtered gastric contents will depend, of couirse, 
upon the amount of pepsin and the amount of free acid present. Artificial 
digestion experiments are at present the only methods by which we may test 
the amount of pepsin. The substances used in these digestion experiments 
are egg-albumin and fibrin. The fibrin may be prepared by beating freshly 
drawn ox-blood with a glass rod until the coagula are distinctly formed. 
These stringy masses are washed thoroughly in water to remove the coloring 
matter of the blood, are then cut into small pieces of uniform size and are 
kept in alcohol for a few days. These hardened masses are placed for one 
to two days in a neutral concentrated solution of carmin. They are then 
washed in water, thoroughly pressed, and are preserved in glycerin to which 
a little carmin has been added. Before being used they should be washed 
in water to remove the glycerin and free coloring matter. The egg-albumin 
is prepared for these experiments by boihng an egg until the albumin is dis- 
tinctly coagulated. This material is then cut into cyhnders of about 5 mm. 
in diameter with a cork borer and are then sectioned into disks i mm. thick. 
These disks may be preserved in glycerin. 

In testing for the presence of pepsin 25 c.c. of gastric juice, which must 
contain free hydrochloric acid, are placed in a flask which contains a few pieces 
of fibrin or a disk of coagulated egg albumin. This flask is then placed in the 
incubator from 37 to 40° and allowed to remain until the protein is completely 
dissolved. If pepsin is present the fibrin will show signs of digestion by swell- 
^See Carlson, Am. Jour. Physiol., 19x5, XXXVIII, 248. 



GASTRIC CONTENTS 79 

ing up in from 15 to 30 minutes, the egg-albumin in from one-half to one hour. 
Within an hour and a half the fibrin should be practically dissolved, while the 
egg-albumin will require about three hours. 

If no hydrochloric acid is present in the gastric juice a few drops of 10 per 
cent, hydrochloric acid are added to 25 ex. of gastric juice and the test per- 
formed in the same manner. A positive result will indicate the presence of 
the zymogen, pepsinogen. 

Quantitative Examination. 

Certain laws have been discovered regarding the action of ferments in 
general and these are applicable to pepsin. Schiitz has found that the rela- 
tive quantities of pepsin in digesting mixtures containing the same quantity of 
hydrochloric acid are proportional to the squares of the quantities of albumin 
digested in the same time, or, in other words, the activity of a ferment varies 
as the square root of its amount. Nirenstein and Schiff ^ have found that this 
law applies only for the less concentrated pepsin solutions. If the quantity of 
pepsin in the digesting fluid is so large that more than 3.6 mm. of albumin 
(see Mett's Test) are digested in 24 hours, the above law does not give reli- 
able values for the quantity of pepsin. The gastric juice under such circum- 
stances must be diluted before this law applies. Sahli rightly calls attention 
to an important reason for diluting the gastric juice before testing for pepsin, 
namely, the constant presence of the products of peptic digestion which in- 
hibit further peptic activity. The gastric juices which contain a diminished 
aniount of hydrochloric acid are the richest in these inhibiting substances and 
should be carefully studied. The presence of these substances gives rise to 
conditions which make it impossible to arrive at accurate conclusions if the 
pepsin value is calculated from the pure gastric juice. 

Hammerschlag's Method. 

Ten c.c. of a i per cent, filtered solution of egg-albumin in 0.4 per cent. 
HCl are poured into two test-tubes. As fresh egg-albumin contains about 13 
per cent, of dry protein, it should be diluted about 13 times to make a i per 
cent, solution. To one of the test-tubes 5 c.c. of gastric juice are added, to 
the other 5 c.c. of distilled water, both being placed in the incubator at body 
temperature for one hour. At the end of this time the albumin in each tube is 
estimated by Esbach's method (see Urine). The difference between the pre- 
cipitate of albumin in the two tubes is equal to the amount of albumin which 
has been digested and forms, therefore, a measure of the peptic activity of the 
gastric juice, the square root of the amount of pepsin being proportionate to 
the quantity of albumin dissolved. This test is open to the objection that the 
albumin of the gastric juice, as well as the albumoses are precipitated by the 
reagent. In one hour not all of the egg-albumin will be digested, normally 
only about 90 per cent. 

Mett's Method. 

This method is, perhaps, the one most frequently used and the one which 

gives quite as accurate results as the others advised. ^ The whites of several 

^ Arch. f. Verdauungskr., 1903, VIII, 559. ' 

2 See Christiansen, Biochem. Ztschr., 1912, XL VI, 257; also, Waldschmidt, Arch. f. d. 
ges. Physiol., i9i2,CXLIII, 189; Hernando and Alday, SigloMedico, igiy.LXIV, ii4andi3o. 



8o DIAGNOSTIC METHODS 

eggs are mixed, in order to avoid accidental variations in the egg-albumin, 
and are filtered. The gas should be removed from this material by the use of 
a suction pump as far as possible. A number of glass capillary tubes, each 
from lo to 30 cm. in length and i to 2 mm. in diameter, are then filled by suc- 
tion with this albumin. They are then laid in the bottom of a vessel which is 
placed for five minutes in boiling water in order to coagulate the albumin. 
The tubes are taken out, wiped carefully, and the ends sealed with paraffin or 
seaKng wax. It occasionally happens that these tubes contain air-bubbles, 
which will, however, disappear in a few days. These longer tubes may be 
kept in stock for a considerable period of time. In performing the test for 
pepsin by the use of these tubes of coagulated albumin, the longer tubes are 
cut into lengths of about 2 cm. and are placed in a small dish or watch-glass 
with 5 c.c. of the gastric juice, which must necessarily be acid in reaction. 
These dishes containing the gastric juice and filled glass tubes are then placed 
in the incubator for 10 to 24 hours. At the end of this time the length of the 
digested column at each end of each tube is measured and the average length 
of the column of albumin digested estimated. The square of this digestion 
length is the measure of the relative amount of pepsin in the gastric juice. 
The unit upon which one may base comparative results of the relative amount 
of pepsin is that quantity of pepsin by which i mm. of albumin in a Mett's 
tube will be digested in 24 hours with an acidity of 0.18 per cent, free HCl. 
The length which theoretically pure pepsin would give is 4 mm., the units rep- 
resented by the pepsin of the gastric juice being anywhere from o to 256. 

Nirenstein and Schiff , for the reasons previously mentioned, advise "the 
dilution of a gastric juice before applying this test, believing that a dilution of 
16 will give more nearly exact quantitative results. Their method is as fol- 
lows: One c.c. of the filtered gastric juice is diluted with 16 c.c. of 0.18 per 
cent. HCl. The procedure is then the same as in Mett's method, the results 
being multiplied, of course, by 16 in order to obtain the actual number of units 
in the gastric juice. In some cases it has been found that a dilution of i to 32 
gives better results, but this is rather unusual. The results of these workers 
show that striking differences exist in individual gastric secretions, figures 
ranging between o and 256 units being obtained. This points to the fact that 
the pepsin concentration is independent of the amount of acid in the gastric 
juice. The quantitative estimation of pepsinogen may be carried out by 
either one of the methods previously outlined for pepsin by rendering the 
gastric juice acid with hydrochloric acid up to i to 2 parts per thousand. 

Method of Thomas and Weber. 

This method^ is based upon the digestion of an acid casein solution in 0.2 
per cent, hydrochloric acid by the gastric juice. The acid casein solution is 
prepared by dissolving 100 grams of finely powdered dry casein in 1,900 grams 
of a solution of hydrochloric acid containing 5.04 grams HCl. Five c.c. of 
gastric juice are added to 100 grams of the casein solution. Dilute with dis- 
tilled water to 250 c.c. and place in the incubator. After the end of one hour 
the digestion mixture is poured into 100 c.c. of 20 per cent, sodium sulphate 

^ Centralbl. f. Stoffwechsel. u. Verdauungskr., 1901, II, 365. 



GASTRIC CONTENTS 8 1 

solution, in which the non-digested casein is completely precipitated. This 
is collected on a weighed filter and washed with distilled water until no trace 
of sulphate reaction is evident. It is then dried with alcohol and ether and 
weighed. The difference in weight between this undigested residue and that 
contained in the original amount taken gives the amount of casein digested. 
This method, as modified by Volhard, consists in the titration of the acid- 
ity of the filtrate from the solution to which the sodium sulphate has been 
added. The total acidity is higher, the more the casein is in the uncoagulated 
form and the increase in the acidity will vary as the square root of the amount 
of pepsin.^ 

(b) Chymosin (Rennin). 

The normal gastric juice contains a second ferment, chymosin, which 
has the function of coagulating milk independently of the presence of acid. 
The zymogen of this ferment becomes active, however, only in the presence of 
acids; that is, the zymogen is converted into rennin by acids. In this process 
of coagulating milk, insoluble casein is formed from the caseinogen of the milk 
by the combined action of the rennin ferment and calcium salts, while the 
curdling of milk is due to the precipitation of unchanged caseinogen by acids. 
It is evident, therefore, that this process resembles very closely that of the co- 
agulation of the blood, as calcium salts are absolutely essential for its success. 
Whether rennin is identical with pepsin, as some believe, is still an unsettled 
question. 2 

Leo's Method. 

Three to five drops of gastric juice are added to 5 or 10 c.c. of fresh un- 
cooked neutral or amphoteric milk and the mixture placed in the incubator 
for 15 to 20 minutes. If rennin is present in normal amounts coagulation will 
be observed. In this process one may not be sure as to whether the curdling 
is due to the action of rennin or to that of the acid. Rennin action occurs 
typically only when no change in the reaction of the milk has taken place. 

RiegePs Method. 

Three to 5 c.c. of neutralized gastric juice are added to 5 to 10 c.c. of 
fresh milk. This mixture is placed in the incubator and left for 15 minutes, 
when distinct coagulation will occur in the presence of rennin. If the milk 
be boiled previous to treatment the result is not so typical. 

Quantitative methods for the determination of rennin are at present 
uncertain and are even of doubtful utiUty. So little is known about the varia- 
tions in the rennin ferment of the gastric juice that an exact determination 
would add little to the clinical history. According to Glassner, pepsin and 
rennin are both diminished in cases of tumor of the fundus, while pepsin is 
diminished and rennin normal in tumors of the pylorus. 

^ For other methods see Jacoby and Solms, Ztschr. f. klin. Med., 1907, LXIV, 159; Fuld 
and Levison, Biochem. Ztschr., 1907, VI, 473; Hata, Ibid., 1909, XXIII, 179; Neilson and 
Bonnot, Arch. Int. Med., 1913, XI, 395; Geselschap, Ztschr. f. physiol. Chem., 1915, XCIV, 
205; Spencer, Jour. Biol. Chem., 1915, XXI, 165; Michaelis, Deutsche Med. Wchnschr,, 
1918, XLIV, 685; Farrington, Lewis and Brown, Jour. Lab. and Clin. Med., 1919, IV, 
635; Northrop, Jour. Gen. Physiol., 1919, II, 113. 

^ See Fuld, Internal. Beitr. z. Path. u. Therap. d. Ernahrungsstor., 1913, V, 104. 
6 



82 DIAGNOSTIC METHODS 

{c) Lipase. 

While it is understood that lipase occurs in the gastric secretion, its 
action is very slight. Normally, gastric digestion is not much concerned 
with the splitting of fat into lower products, but, as Volhard has shown, 
this action does occur. In testing for the presence of lipase in gastric con- 
tents it is necessary that the examination be made after the stomach is thor- 
oughly washed out following the administration of a test meal free from fat. 

This ferment may be detected qualitatively by adding a small piece of 
fresh neutral butter to the gastric juice and placing the mixture in the incu- 
bator for one hour. At the end of this time a distinct odor of butyric acid 
will be observed. 

It may be said that owing to this lipolytic action statements have arisen 
that the Sahli test meal gives erroneous results Careful work by Seller 
shows that the amount of fat decomposed under the conditions of the test 
meal is so slight that it may be neglected. Volhard found that after two 
hours from 30 to 36 per cent, of the fat was split up into fatty acids, which 
aid in dissolving the bile and in forming an emulsion with the neutral fat in 
the intestinal canal. 

The Products of Protein Digestion. 

It is generally stated that the action of the pepsin and hydrochloric acid 
of the gastric juice upon protein material passes through the following stages: 
The albumin is first conyerted into acid-albumin (syntonin), then into 
albumoses, of which there are four (prot-albumose, hetero-albumose, dys- 
albumose, and deutero-albumose), and ultimately into peptone. It is well 
established that pepsin itself will not carry the hydrolysis beyond the stage 
of peptone or somewhat lower polypeptids. The finding of amino-acids and 
hexon bases, by the author as well as by others, in the contents of a dilated 
stomach must be interpreted as meaning a conversion of the pepsin products 
into lower cleavage units through the action of some other ferment, whether 
derived from the intestinal canal or excreted into the stomach from a malig- 
nant growth. Pepsin has, therefore, a function distinct from that of trypsin, 
attacking different linkings within the protein molecule. It is practically 
never necessary to test the stomach contents for such material in clinical 
work, so that the writer will refer to text-books on physiologic chemistry. 

The Products of Carbohydrate Digestion. 

The pure gastric juice, owing to its acid content, inverts sugars to a certain 
extent. The amount of this inversion depends on the number of free hydro- 
gen ions arising from the acid, and is only very slight under normal conditions. 

As previously described, the saliva converts starch into soluble substances 
through the stage of soluble starch, erythrodextrin, achroodextrin and finally 
maltose. The action of the ptyalin of the saliva is inhibited by the free acid 
of the gastric juice, but the action of this salivary ferment is so rapid that 
from 50 to 75 per cent, of the starch is converted into a soluble form.^ While 
these products are not ordinarily tested for in the gastric contents, the fact 
of their formation and presence must be remembered. An excess of starchy 

^ See Maxwell, Biochem. Jour., 1915, IX, 323. 



GASTRIC CONTENTS 83 

material in the food will lead to an increased amount of such decomposition 
products in the stomach when the acid of the gastric juice is not present in 
sufficient amount to inhibit the action of the ptyalin of the saliva. 

Blood. 

Blood is not a normal constituent of the gastric contents, but is found fre- 
quently in conditions associated with erosion, in ulcer, and in carcinoma. 
The appearance of the blood in cases of ulcer is usually that of fresh bright 
red blood, which may, however, be changed to a brownish substance due to 
the action of the excess of acid commonly present in this condition. In car- 
cinoma the blood is more intimately mixed with the stomach contents and 
appears in the form of brownish-black clumps, constituting the so-called 
^'coffee-ground" material. The tests for the presence of blood will be given 
under Feces. ^ 

Gases. 

The stomach usually contains a certain amount of gas which may have 
been swallowed, may have passed into the stomach from the duodenum, or 
may have been produced in the stomach by processes of fermentation. The 
examination for these gases is not of great clinical importance, but a general 
idea of the different kinds of gases present seems essential. During the proc- 
esses of normal digestion, nitrogen, oxygen, and carbon dioxid may occur from 
the protein digestion, while hydrogen, marsh-gas, and olefiant gas may arise 
from the carbohydrate hydrolysis. In abnormal processes of digestion we 
may find ammonia and hydrogen sulphid arising from decomposing protein 
material. The work of Boas seems to indicate that the hydrogen sulphid is 
more commonly present in cases of benign gastric dilatation and is rare in car- 
cinoma. This hydrogen sulphid does not seem to be produced either in the 
presence of free hydrochloric or of lactic acid. The presence of hydrogen sul- 
phid can, however, not be considered as a specific substance in the stomach 
contents, as Dauber has shown that almost every stomach contains bacteria 
which may produce this gas from sulphur-containing bodies. In cases of dila- 
tation of the stomach, providing the motility be sufficiently diminished, we 
find fermentation with resulting gas production even though hydrochloric 
acid be present. Such a condition never occurs if the motility is normal, 
utterly regardless of the amount of hydrochloric acid, as in cases of diminished 
hydrochloric acid lactic acid will usually prevent such a process. 

One may show the presence of the gases in the stomach contents by filling 
a fermentation tube with the well-mixed unfiltered gastric contents and plac- 
ing it in the incubator for some time. If there is no gas within 24 hours it 
may be wise to wait at least 48 hours to permit of the proper diffusion of the 
gas. If gas is formed its nature may be determined by the ordinary chemical 
tests. This test has some value in determining the degree of stagnation of 
the stomach contents, but it must be remembered that a small amount of 
gas is contained in the normal stomach. 

Other substances, such as acetone, have been found in the gastric contents 
in pathologic conditions, but tests for these substances are rarely of impor- 
^ See Leviton, Jour. Lab. Lab. and Clin. Med., 191 6, I, 761. 



84 DIAGNOSTIC METHODS 

tance. In conditions associated with the presence of acetone in the stomach 
contents this substance is usually detected in the breath. 

Functions of the Stomach and its Contents. 

The stomach is to be regarded as a specialized portion of the alimentary 
tube in which the first stages of digestion of protein material take place. 
This occurs under the combined action of the hydrochloric acid and pepsin, 
the resulting products being gradually passed into the duodenum through the 
pylorus. An increased acidity of the gastric juice may be associated with a 
distinct spasm of the pylorus so that food material cannot pass into the duo- 
denum. On the other hand, a lessened degree of acidity is associated with 
hypermotility, the contents passing rapidly into the intestine, where it i& 
acted upon by the pancreatic ferments. Beside the function of digestion, 
principally of the protein foods, the stomach serves as a reservoir to hold the 
food material, allowing it to pass only in small portions into the bowel at any 
one time.^ Owing to the presence of hydrochloric acid, the gastric juice is 
antiseptic, rendering inert many but not all types of bacteria. ^ Moreover, the 
hydrochloric acid activates the zymogens and thus permits of action upon all 
types of food material. The work of Pawlow has shown that the acid of the 
gastric juice is one of the most powerful stimulators of pancreatic secretion. 
The mechanism of correlation between the stomach and bowel is more easily 
understood if this point be borne in mind. The acid chyme coming from the 
stomach is poured out only in small portions at a time so that the pancreatic 
juice secreted may act upon the smaller portions as they are passed into the 
bowel. 

VI. Motility of the Stomach 

It is probably true that disturbances in the motility of the stomach are in 
reality of more importance than are those in the secretory activities.^ Under 
normal conditions of motility the food material passes into the intestine and 
is digested there, although no previous gastric digestion has taken place. If 
the motility be much impaired, stagnation of food with resulting dilatation of 
the stomach will occur, which will give rise to more or less serious disturbance. 
The motor disturbances are of three types, (i) vomiting, (2) hypermotility, 
and (3) motor insufficiency. The most important of these is the latter, as 
the former has Kttle influence upon actual digestion in the stomach, although 
the patient may suffer for want of adequate nutrition; while in hypermotility 
the gastric disturbance will not be much noticed, owing to the fact that the 
food is rushed into the duodenum where it is digested. The consequences of 
motor insufficiency may be either disorders of secretion, decomposition, or 
both. Simple pathologic conditions which hinder the emptying of the stom- 
ach, such as ulcers, cicatrices, spasm of the pylorus, and simple atony, are 

1 Tangl and Erdelyi (Biochem. Ztschr., 1911, XXXIV, 94) and Fejer (Ibid., i9i3,LIII, 
168) have shown that the fats are discharged into the bowel at different rates depending 
upon their melting point and their viscosity. The higher the melting point the longer a fat 
remains in the stomach, while the more viscid a fat the less does it retard the discharge of 
non-fat foods. 

2 See Gregersen, Centralbl. f. Bakteriol., 1916, LXXVII, 353; Scheer., Arch. f. Hyg., 
1919, LXXXVIII, 130. 

3 See Carlson, Am. Jour. Physiol., 1912, XXXI, 151. 



GASTRIC CONTENTS 85 

usually associated with hyperacidity, while malignant conditions usually 
show a diminished secretion.^ It is quite rare to find a case of motor insufii- 
ciency without disorder of secretion, rarer at all events than disorders of se- 
cretion without motor disturbances (Schmidt). Motor insufficiency is quite 
commonly followed by decomposition of the gastric contents and may even 
be considered the chief cause of such decomposition. Under these conditions 
of disturbed secretory and digestive activity associated with motor insuffi- 
ciency we find the absorptive power of the stomach very much affected. 

Hypermotility is seen in many cases of hyperacidity, but it must be 
remembered that primary hyperacidity may cause spasm of the pylorus and 
hence bring on a distinct motor insufficiency. We cannot, therefore, judge of 
the motility of the stomach from the degree of acidity of the gastric con- 
tents. An enlarged stomach is not necessarily associated with motor in- 
sufficiency. Cases of megalogastria are more or less frequent in which the 
motility is practically normal. When a dilatation is associated with motor 
insufficiency it is clinically styled an ectasia or ectasis, being known as atonic 
gastric ectasis if the condition is due to weakness of the muscle, while it is 
styled hypertonic gastric ectasis if due to pyloric stenosis. Normally, no 
food should be found in the stomach within seven to eight hours after taking, 
no matter how large the meal. ^ 

Leube^s Method. 

Leube administers a Riegel test meal and washes out the stomach with a 
liter of water six hours after. If only very slight traces of food are found in 
the washings the motor power is regarded as normal. 

Boas' Method. 

Boas administers a simple evening meal consisting of meat, bread and 
butter, and tea, washing out the stomach the following morning. If any food 
material is found the motor insufficiency is considerable. If the stomach be 
washed out previous to the administration of the evening meal no food should 
be found in the stomach in the morning. 

Method of Ewald and Sievers. 

This test is based upon the observation that salol is decomposed into car- 
bolic and salicylic acids only in an alkaline medium. As the salicylic acid is 
eliminated in the urine in the form of salicyluric acid, it is possible to de- 
termine the rate of passage of salol from the stomach to the small intestine. 
It seems necessary to state that the assumptions on which this test are based 
are partially wrong. In the first place salol is split into its constituents by 
gastric juice within 15 minutes, although the degree of dissociation is slight. 
Moreover, a certain amount of absorption of salicyclic acid takes place from 
the stomach so that a reaction may be obtained in the urine within 15 minutes 
in cases in which no hypermotility exists. 

One gram of salol is given to the patient immediately after a meal. The 

urine is then collected every 1 5 mi-nutes for two hours and tested by the addi- 

^ See Hamburger and Friedman, Arch. Int. Med., 1914, XIV, 722. 
2 See Faulhaber, Berl. klin. Wchnschr., 1914, LI, 1355; Levy and Kantor, Arch. Int. 
Med., 1916, XVII, 476. 



86 DIAGNOSTIC METHODS 

tion of a small amount of ferric chlorid solution, which will give a violet 
color in the presence of salicyluric acid.^ 

Under normal conditions, according to Ewald, a positive reaction occurs 
in from 45 to 75 minutes. A further delay above 75 minutes is indicative of 
motor insufficiency, the degree of insufficiency bearing some relation to the 
time of appearance of this reaction. Should no result be obtained after 24 
hours a stenosis of the pylorus is highly probable. 

As the writer has so frequently found a reaction for salicyluric acid in the 
urine within 15 minutes, which is due not to the action of the hydrochloric 
acid or the ferments of the gastric juice, but to the moisture, temperature, and 
bacteria, he is accustomed to use the time at which a reaction for this sub- 
stance disappears from the urine rather than the time at which it makes its 
first appearance, as the basis of judgment regarding the motility of the 
stomach. Normally, no reaction for salicyluric acid should occur in the 
urine after 24 hours although Huber states that it may take 26 to 27 and hence 
limits his time to these latter figures. It might be wise to follow the sugges- 
tion of Sahli and determine both the time of appearance and disappearance. 

Sahli has called attention to the fact that we are not justified in assuming 
a pyloric stenosis in case food material is found in the stomach even several 
days after being taken. He adds that the emptying of the stomach is regu- 
lated by the intestine rather than by the stomach itself, since nutritive sub- 
stances reaching the intestine effect a reflex closure of the pylorus (von 
Mering's reflex) until the intestine has completed its work. The motor ac- 
tivity of the stomach should, therefore, be examined under conditions in 
which this reflex does not occur. This he determines by estimating the length 
of time required by the stomach to empty itself of a half-liter of water, the 
stomach being thoroughly washed out previously.^ 

Winternitz Test. 

Winternitz has recommended the use of iodipin instead of salol for testing 
the motility of the stomach. This substance is not affected by the gastric 
contents, but is acted upon in the intestine by the pancreatic secretion and 
bile in such a way that iodin is set free. This may be tested for in the 
saliva by adding to it a little starch paste, w^hen a distinct blue color will be 
observed within 15 to 45 minutes. 

VII. Absorptive Powder of the Stomach 

The absorptive powr of the stomach is not of great importance clinically, 
as the greatest part of absorption occurs from the intestinal tract. However, 
tests for such power have a certain associated value and are, therefore, usually 
made. For this purpose Penzoldt and Faber advance a method depending 
upon the principle that under physiologic conditions potassium iodid is rap- 
idly absorbed by the gastric mucous membrane and is immediately eliminated 

^ Thoburn and Hanzlik, Jour. Biol. Chem., 1915, XXIII, 163, outline methods for the 
quantitative determination of this acid in urine. • 

2 See Boas (Deutsch. med. Wchnschr., 1912, XXXVIII, 455), Kemmerling (Arch. f. 
Verdauungskr., 1914, XX, 49) and Wartensleben (Ibid., 66) for a discussion of the Boas, 
chlorophyl test. 



GASTRIC CONTENTS 87 

in the saliva. A capsule containing two* to three grains of potassium iodid is 
given to a patient shortly before a meal. The saliva is then tested as follows 
for the presence of potassium iodid at intervals of two to three minutes. The 
saliva is slightly acidified with nitric acid and treated with a few drops of 
starch paste when the characteristic blue color of iodid of starch will be formed 
by the action of the iodin liberated from the potassium iodid by the nitric 
acid. Under physiological conditions the first trace of iodin will appear in the 
saliva within ten minutes of its administration upon an empty stomach. 
Under pathological conditions a delayed reaction may be observed in almost 
all diseases of the stomach, especially in dilatation and in carcinoma. The 
test will naturally be delayed in case the stomach is filled with food. This test 
has little value, as it may appear or not in all types of gastric disease. Von 
Mering has found that potassium iodid is not absorbed at all from the stom- 
ach even within two or three hours, so that the iodin appearing in the saliva 
may be due to absorption from the intestine.^ 

VIII. Indirect Examination of the Stomach Contents 

As not all cases of disease of the stomach permit of examination by re- 
moval of the contents through the stomach-tube, methods have been ad- 
vanced to permit of indirect determination as to the activity of the stomach 
contents. These methods do not permit of accurate determination of the 
acidity or of the ferments of the juice, but do give much information regarding 
the normal digestive powers and motility of the stomach. 

Giinzburg's Method. 

A tablet of o . 2 gram of potassium iodid is placed in a piece of the thinnest 
possible strongly vulcanized rubber tubing measuring about 2.5 cm. in length. 
The ends of the tubing are folded and the package tied with three threads of 
fibrin which have been hardened in alcohol. The package is now tested by 
placing it in warm water for several hours and examining the water for potas- 
sium iodid. The patient swallows one of these packages three-quarters of 
an hour after an Ewald meal, the saliva being tested for potassium iodid at 
intervals of 15 minutes. In the presence of free hydrochloric acid in normal 
amounts the threads of fibrin are dissolved and the potassium iodid is ab- 
sorbed, giving a reaction in the saliva in from one to one and three-quarters 
hours. In cases of hypochlorhydria the reaction is delayed, a delay of six 
hours indicating a practical absence of free hydrochloric acid. 

This test very frequently gives rehable results, but the threads of fibrin 
soon become brittle and break on swallowing the package so that a reaction 
for potassium iodid under these conditions would have no value. 

Sahli's Desmoid Reaction. 

Sahli has recently introduced the ^^ Desmoid bag^' for use in estimating the 

functional activity of the stomach. These bags are made of the ordinary 

rubber-dam used by dentists and contain a pill of o . 05 gram of methylene 

blue and o . i gram of iodoform. The bag is tied, in a manner especially 

1 See Mendel and Baumann (Jour. Biol. Chem., 1915, XXII, 165), who show that ab- 
sorption is not a prominent function of the stomach. 




88 DIAGNOSTIC METHODS 

outlined by Sahli, with cat-gut which has been allowed to dry but has been 
untreated chemically. This gut, according to Sahli, is digested only by the 
gastric juice and not by the pancreatic juices. This pill is administered to 
the patient immediately following the noon meal and the urine and saliva 
tested at iiitervals of one hour, beginning three hours after administration of 
the pill. The digestion of the gut by the gastric juice liberates the pill and 
permits of the absorption of both the methylene blue and the iodoform. The 
methylene blue will appear in the urine coloring it green within six hours, 
while the iodin will be found in the saliva within two hours. Should the color 
of the urine not be distinctly green, this tint may be more clearly brought 
out by adding a few drops of acetic acid and boiling. 
Variations from the periods indicated above denote a 
hyperacidity or a hypoacidity of the gastric juice accord- 
ing as the time of appearance of the reactions is lessened 
or increased. As the gut is digested only by the gastric 
juice^ a non-appearance of either reaction would indicate 
an anachlorhydria. 

The writer has used these desmoid bags in a large num- 
F R _s hi'' ^^^ ^^ cases and has found them fairly reliable, giving re- 
Desmoid bag. suits which have, in many cases, been confirmed by chem- 
ical analysis. As these bags are not obtainable in the mar- 
ket, he has been forced to make them himself and has found that the technic of 
Sahli must be followed very closely, especially as regards the tying of the gut. 
Other methods, such as those of Dunham, Turck, and Einhorn, have been 
advocated, but possess no advantages over those outlined.^ 

IX. The Gastric Juice in Disease 

(i) Hj^erchlorhydria. 

By hyperchlorhydria is meant the secretion of an abnormally acid gastric 
juice whose acidity is due to an excess of free hydrochloric acid. This secre- 
tion is much more marked during digestion, being less frequent on an empty 
stomach. Under these conditions we usually find an increased total acidity 
along with the increase of free hydrochloric acid. A condition which is char- 
terized by a high total acidity with a very high amount of organic acid would 
not, of course, be considered in this connection. A hyperacidity or hyper- 
chlorhydria exists when we have more than o . 2 per cent. (60°) of free hydro- 
chloric acid (see p. 69). 

This condition may be due to pathologic changes in the mucosa or to direct 
nervous influences. Cases of pure hyperchlorhydria are occasionally very 
stubborn and may be associated with almost any variety of abnormal gastric 
function. While we find hypermotility of the stomach in many cases of hyper- 
chlorhydria, we very frequently note a diminished motility due to spasm of 

^ Gregersen (Arch, f, Verdauungskr., 19 13, XIX, 43) advocates the use of Schmidt's test 
of administering 100 grams of very slightly cooked meat and the later study of the stools 
for connective-tissue fiber. 

2 See Friedrich, Berl. klin. Wchnschr., 1913, L, 32; also, Rehfuss, Am. Jour. Med. Sc. 
1914, CXLVII, 848; Einhorn, Jour. A. M. A., 1919, LXXIII, 1509. 



GASTRIC CONTENTS 89 

the pylorus. This condition brings about a stagnation of the stomach con- 
tents and a consequent increase in the fermentative processes. The acidity 
in such cases may run as high as 200° or over and the digestive powers of the 
gastric juice, as regards protein substances, may be much increased, the car- 
bohydrate digestion being correspondingly diminished. These facts point to 
the reason for the administration of an increased protein diet in such cases, 
the protein combining with the hydrochloric acid and thus taking a portion of 
the excess from the field of action. 

While this condition is not a distinct entity, yet we find many cases which 
come under the heading of idiopathic hyperchlorhydria and which are not 
associated with other pathologic conditions. Some of these cases are purely 
functional and clear up promptly under proper treatment, while others are of 
nervous origin and are remedied only when the etiologic factor is eliminated. 
In this latter type of hyperchlorhydria the degree of acidity varies with the 
nervous symptoms, giving rise to the term " heterochylia.^^ 

(2) Hypersecretion (Gastrosuccorrhea). 

By hypersecretion is meant an excessive secretion of gastric juice which 
is out of proportion to the physiologic stimulus. This hypersecretion occurs 
even when no stimulus is present, is always pathological, and, according to 
Riegel, always produces pathological results. A hyper- or continuous secre- 
tion may be determined by finding a fairly large amount of gastric juice in the 
fasting stomach under conditions which rule out stenosis and stagnation. The 
stomach is washed out before the patient retires, the contents being with- 
drawn the following morning. If a quantity of highly acid fluid is obtained, a 
hypersecretion is proven. The quantity taken from the fasting stomach 
should never be more than 100 c.c. This secretion, to be called a hypersecre- 
tion, must contain no food remnants, no sarcinae nor yeast cells, but should be 
distinctly acid. 

This condition is probably a functional neurosis, being constant or inter- 
mittent and a part of a general neurosis, a secretory neurosis, or the result of 
organic nervous disease, such as the gastric crises of tabes dorsalis. Reich- 
mann has reported cases of the periodic or intermittent type, during the inter- 
vals .between the attack the digestion of the patient being normal. Such cases 
are known as Reichmann's disease. 

The chronic cases are of long duration and have a gradual onset. The 
patient complains of much discomfort, feeling of weight or depression in the 
stomach, pain during digestion, vomiting, especially at night, and a gastric 
contents with a large amount of free hydrochloric acid. Dilatation of the 
stomach sooner or later comes on as the result of spasm of the pylorus ind uced 
by the hyperacidity. In these dilated stomachs we find, of course, products 
of fermentation and many yeast cells and sarcinae. 

(3) Achylia Gastrica. 

This condition may arise either from a functional disturbance of the 
mucosa or a true atrophy of the mucosa. This latter state, known as atrophic 
gastritis, may be the end stage of a chronic gastritis or the result of carcinoma. 



go DIAGNOSTIC METHODS 

When this condition is not due to direct gastric disturbance, it is more fre- 
quently seen in connection with pernicious anemia, in which the general nutri- 
tion is very much below par. The local condition may not be suspected, as 
the not uncommon hypermotility, may prevent attention being drawn to 
the stomach. 

For a diagnosis of achylia, the test meal of Ewald gives very good results. 
Examination of the gastric contents shows that the food is little changed, the 
total acidity very low (i to 6°), no free hydrochloric acid, gastric ferments 
much diminished or entirely absent, and lactice acid only in small amounts. 
The motility of the stomach is usually little impaired, so that the retention of 
food is unusual.^ 

(4) Acute Gastritis. 

The stomach contents of acute gastritis shows a diminished total acidity, 
little or no free hydrochloric acid, organic acids relatively increased, much 
mucus and undigested food. This condition is usually brought on by direct 
irritation and is generally easily remedied by total abstinence. The material 
for chemical examination is usually obtained in these cases from the vomitus, 
as the passing of the stomach-tube is very rarely tolerated. 

(5) Chronic Gastritis. 

All grades of this condition may exist up to complete atrophy of the 
mucosa. Examination of the stomach contents shows practically no diges- 
tion of the food material, much mucus intimately mixed with the food parti- 
cles, the secretion usually diminished, free hydrochloric acid diminished or 
absent, ferments much reduced, protein digestion small, starch digestion little 
affected, microscopic examination showing the presence of many epithelial 
cells and leucocytes. There are some cases of chronic gastritis in which a 
hyperacidity of the juice is evident, but these are rare, the usual finding being 
one of diminished acidity. The motility of the stomach in these cases is 
sometimes normal, sometimes increased, or may be diminished. One of the 
most characteristic findings in this condition is the presence of a large amount 
of mucus containing either leucocytes or their nuclei and epithelial cells from 
the walls of the stomach. If there is little acid present in the gastric contents 
the mucus may swell up and appear greater in volume. As a general rule, it 
may be said that the mucus and the hydrochloric acid vary inversely as their 
amounts. 

(6) Nervous Dyspepsia. 

This condition of nervous dyspepsia is part of a general neurosis and may 
show no characteristic findings in the stomach contents. The degree of 
acidity may range from a normal to either a hyper- or a hypoacidity, while the 
amount of ferments present will not usually vary. The findings in nervous 
dyspepsia are not at all constant, varying at different examinations. We have, 
therefore, more or less distinct methods of differentiation between this con- 

1 Disque (Arch. f. Verdauungskr., 1914, XX, 366); Orloff (Russk Vrach., 1914, XIII, 
1006) believes there eventually occurs a vicarious increased secretion of trypsin in this 
condition. See, also, Rehfuss, Am. Jour. Med. Sc, 1915, CL, 72; Andresen, Med. Record, 
i9i6,LXVII, 1629; Beck and McLean, Southern Med. Jour., 1919, XII, 594. 



GASTRIC CONTENTS 9 1 

dition and chronic gastritis. In the first place, the acidity of chronic gastritis 
remains constant for several examinations while that of nervous dyspepsia is 
variable. The ferments are diminished in cases of gastritis, while they are 
normal in nervous dyspepsia. Much mucus is found in gastritis while little 
or none is present in dyspepsia. The cases of nervous dyspepsia are partially 
associated with distinct errors in eating. The influence of nervous conditions 
over gastric function has been very well expressed by Emerson when he says, 
"a neurasthenic will often worry his subliminal gastric sensations into the 
sphere of consciousness." 

(7) Ulcer of the Stomach. 

The diagnosis of ulcer of the stomach depends, to a large extent, upon the 
clinical symptoms of the disease rather than upon the examination of the 
stomach contents.^ While the symptoms of this disease, increasing dyspepsia, 
pain, vomiting, blood in the vomitus, and hyperacidity of the vomitus, are well- 
known, the stomach-tube should rarely ever be used in obtaining the con- 
tents of the stomach, owing to the danger of perforation in such cases. ^ 

The vomitus of such cases is usually ejected from one to three hours after a 
meal and contains well-digested food. Blood mayor may not be present and 
may be either fresh red blood or dark in color from the formation of hematin. 
The total acidity of the gastric contents is usually increased, hydrochloric acid 
constituting a large part of this total acidity. A single examination of the 
gastric contents will rarely determine anything about an ulcer, so that re- 
peated examinations of the vomitus must be made to obtain a general idea of 
the acidity. In these cases blood is usually present in the feces and may be 
detected as outlined later. ^ When an ulcer is complicated by a beginning car- 
cinoma, we may find all types of variation in the acidity of the stomach 
contents. . 

(8) Carcinoma of the Stomach. 

In no other condition of the stomach is an absolutely certain early diagno- 
sis to be so much desired as in carcinoma of the stomach. The chemical 
features of the gastric juice in this condition may be very suggestive or may 
be negative. The clinical history of the case along with the age of the patient 
are probably of more importance in making a diagnosis of carcinoma than are 
variations in the chemical composition of the juice. The local symptoms of 
carcinoma of the stomach are sometimes as variable as are the changes in 

1 See Verbrycke, Am. Jour. Med. Sc, 1913, CXLVI, 742. 

2 Rosenow (Jour. Am. Med. Assn., 1913, LXI, 1947) has shown the close relationship of 
streptococcic invasion to gastric ulcer. See, also, Rosenow and Sanford, Jour. Infect. Dis., 
1915, XVII, 219; Rosenow, Ibid., 1916, XIX, 333; Bolton, Jour. Path, and Bacteriol., 
1915, XX, 133; Wilensky and Geist, Jour. A. M. A., i9i6,LXVI, 1382; Steinharter. Boston 
Med. & Surg. Jour., 1916, CLXXIV, 678; CLXXV, 59; 1917, CLXXVI, 461; Hardt, Am. 
Jour. Physiol., 1916, XL, 314; Celler and Thalhimer, Jour. Exper. Med., 1916, XXIII, 
791; Dragstedt, Jour. A. M. A., i9i7,LXVIII, 330; Smithies, 111. Med. Jour., 191 7, XXXI, 

149- 

3 See Lichty, Am. Jour. Med. Sc, 1914, CXLVIII, 680; Smith, Jour. Med. Research, 

1914, XXX, 147; Smithies, Arch. Diagnosis, 1915, VIII, 147; Interstate Med. Jour., 1915, 
XXII, 207; Boas, Arch. f. Verdauungskr., 1915, XXI, 94; Sippy, Jour. Am. Med. Assn., 

1915, LXIV, 1625; Mayo, Ibid., 1915, LXV, 1069; Cheney, Ibid., 1227; Stone, Jour. 
A. M. A., 1916, LXVI, 324;Dahl,Hygiea, i9i6,LXXVIII. 1408; Moschcowitz, Am. Jour. 
Med. Sc. 1916, CLII, 714. 



92 DIAGNOSTIC METHODS 

the gastric juice, so that every possible point in diagnosis should be taken 
advantage of, if for no other purpose than to exclude this condition. ^ 

Perhaps the most important sign of carcinoma is the absence of free hydro- 
chloric acid. Although this condition is present in about 85 per cent, of cases, 
it cannot always be traceable to carcinoma, as it may occur in atrophic gastri- 
tis and advanced chronic gastritis. This lack of free hydrochloric acid is due 
to the union of this acid with some body which in itself does not show an 
alkaline reaction. Von de Velden suggests that the secretion from the cancer 
is the active agent in neutralizing the hydrochloric acid. Moreover, the pro- 
ducts of protein digestion might have some power in neutralizing the acid, 
Emerson having shown that hexone bases are present as a result of the action 
upon the protein of a ferment^ derived from the tumor itself. Certainly, in 
cases of carcinoma the total nitrogen of the stomach contents is much increased, 
so that such bodies are probably a very great factor in the diminution of the 
hydrochloric acid. This reduction in hydrochloric acid is also influenced by 
changes in the mucosa to such an extent that the active secretion is diminished. 
The failure of free hydrochloric acid is usually a very early symptom, but it 
must be remembered that hydrochloric acid may be present in normal amounts 
or even in increased amounts when the carcinoma is small and occupies the 
pyloric region or when this growth develops on the base of an old ulcer. ^ The 
acidity may vary markedly from day to day, sometimes showing no free hydro- 
chloric acid and sometimes a considerable amount. This variation is of con- 
siderable practical importance. In cases of carcinoma of the esophagus the 
disappearance of hydrochloric acid from the gastric contents seems necessarily 
to be the result of the secretion of the tumor neutralizing the gastric juice. 
Along with this diminution in free hydrochloric acid the total acidity is also 
diminished.^ 

The presence of an increased amount of lactic acid is a very valuable sign 
in cancer.^ About 90 per cent, of patients show the presence of lactic acid 
when there is no free hydrochloric acid, but when there is a large amount of 
combined HCl pointing to a free secretion of this acid. Lactic acid may not 
be present in cases of carcinoma, especially those in which the growth is upon 
the base of an old ulcer, or it may be present in conditions other than carci- 
noma, such as chronic gastritis, associated with atrophy of the mucosa and 
dilatation of the stomach, especially when a benign stenosis of the pylorus 
exists. It must be stated, however, that we usually find an increased secre- 
tion of hydrochloric acid in cases of simple benign pyloric stenosis, so that 
lactic acid cannot be formed in the presence of this increased hydrochloric 
acid. According to Riegel, the chief cause of the lactic acid formation is the 

^ See Schiitz, Wien. klin. Wchnschr., 1913, XXVI, 1053, and Arch. f. Verdauungs- 
Kr., 191S, XXI, 421; Patella, Rif. Med., 1916, XXXII, i, 29, 57 and 85; Kahn, Jour. Lab. 
and Clin. Med., 1916, II, 103. 

2 Kriloflf (Med. Obozr., 1913, LXXIX, i) believes trypsin is always present in cancer, 
especially of the pylorus. See, also, Halpern, Mitt. a. d. Grenzgeb. der Med. u. Chir., 1914, 
XXVIII, 709. Pron, Compt. rend. soc. biol., 1916, LXXIX, 68. 

^ See Levy, Beitr. z. klin. Chir., 1914, XCIII, 696; Ochsner, Jour. Am. Med. Assn., 1915, 
LXV, 1073; Zoeppritz, Mitt. a. d. Grenzgeb. d. Med. u. Chir., 1916, XXIX, I. 

* See Friedenwald, Am. Jour. Med. Sc, 1914, CXLVIII, 660; Smithies, Jour. Am. Med. 
Assn., 1914, LXIII, 1839; Ibid., 1915, LXIV, 643. 

^ See Schryver and Singer, Quart. Jour. Med., 1913, VI, 71 and 309. 



GASTRIC CONTENTS 93 

combination of motor insufficiency with a hypoacidity associated with a 
diminished secretion, both of acids and ferments. This diminution in the 
amount of ferments is in no way specific for cancer, as it is in reaUty due to 
the chronic gastritis which is set up by the tumor. As lactic acid cannot be 
formed in the presence of an appreciable amount of free HCL, we do not al- 
ways find after a test meal a large excess of this acid, especially in those cases 
in which considerable combined HCl is present. It seems advisable, there- 
fore, in testing for the presence of lactic acid, to examine the contents of the 
fasting stomach in the morning after it has been well washed out the pre- 
ceding evening. Whether we are to assume that lactic acid is formed by the 
action of the organisms in the stomach upon food material, whether it be a 
normal product of digestion, or whether it is a product of the activity of a 
ferment derived from the tumor, must be left for future investigation. 

The vomited material or the material obtained by washing out the stom- 
ach shows very little digestion of the protein elements, while the carbo- 
hydrates are well hydrolyzed. The amount of material in the stomach will 
be large or small depending upon the degree of stenosis, so that it is nothing 
unusual to obtain several pints of material containing undigested protein 
residue. Microscopic examination of this material may show cellular masses 
washed off from the tumor or may show these fragments embedded in masses of 
blood. ^ Blood is a usual finding in cases of carcinoma and may be detected 
by the methods discussed under Feces. Sarcinae and yeasts are rare, the 
former occurring more frequently in cases of marked dilatation. The Boas- 
Oppler bacilli are more or less constant findings in carcinoma, occurring in 
about 90 per cent, of the cases and only very rarely in any other condition. 
Salomon's Test. 

This test is based upon the fact that albumin is secreted from the car- 
cinoma itself and passes into the gastric contents. The patient is placed 
upon an absolutely protein-free diet for 24 hours and the stomach carefully 
washed out at the end of this time with 400 c.c. of physiological salt solution. 
A few hours thereafter the contents of the stomach are removed and the remain- 
ing material washed out with 400 c.c. of physiological salt solution. The total 
nitrogen and the albumin are then estimated, the former by the Kjeldahl 
method and the latter by the Esbach method (see Urine). The nitrogen 
ranges from 10 to 70 mg. per 100 c.c. in cases of carcinoma, while in other 
conditions it varies from o to 16 mg. per 100 c.c. The Esbach reaction gives a 
distinctly appreciable precipitate for albumin, anything over 0.5 parts per 
1000 being considered indicative of carcinoma. While this test is not infallible, 
yet the writer has found it present in so many cases of carcinoma that he is in- 
clined to make a very strong presumptive diagnosis on the basis of this test.^ 
Neubauer and Fischer's Test. 

These investigators,^ working in Mliller's clinic, have taken advantage 
of the well-known fact that normal peptic digestion does not proceed to the 

1 See Simon and Caussade, Presse med., 1914, XXII, 265; Loeper and Binet, Arch, des 
Mai. de I'App. digestif, 1914, VIII, 181; Caussade, Rev. de Med., 1914, XXXIV, 428. 

2 See Hohlbaum, Arch. f. klin. Chir., 1914, CIV, 1069. 

3 Deutsch. Arch, f . klin. Med., 1909, XCVII, 499. 



94 DIAGNOSTIC METHODS 

formation of amino-acids but stops at the peptone or peptid stage. Further, 
it has been shown by Emerson, Fischer and others that digestion of protein 
goes somewhat further in carcinomatous stomachs than in normal ones, owing 
to the fact that carcinomatous tissue, as well as various body tissues, contains 
proteolytic ferments capable of producing more complete hydrolysis than does 
pepsin (see Lewin^). Neubauer and Fischer, following the investigations of 
Erdmann and Winternitz, Glaessner, and Volhard, use this hydrolysis of 
peptids by such proteolytic ferments for diagnostic purposes. Their method 
is as follows : The contents of the stomach are withdrawn one-half to three- 
fourth hour after an Ewald meal; lo c.c. of the filtered material are mixed with 
a little gylcyl-trytpophan^ and a little toluol is added to prevent bacterial ac- 
tion. The mixture is placed in the incubator at 37°C. for 24 hours. At the 
end of this period, withdraw 2 or 3 c.c. of the mixture from beneath the toluol 
layer, place this in a test-tube, add a few drops of 3 per cent, acetic acid and 
then allow the fumes of bromine to pass into the tube from an open bottle of 
bromine. This part of the technic must be carried out very carefully as an 
excess of bromine will obscure and even destroy the reaction. After shaking 
the tube a positive reaction is shown by the appearance of a rose-red or intense 
red color. If no red color appears, carefully add more brom'ne vapor and 
shake. This addition may be continued until an excess of bromine is evi- 
denced by a light yellow color of the mixture. 

These workers have shown that errors may arise in this test due to the 
presence of (i) tryptophan in the gastric contents, (2) peptid-splitting bacteria, 
(3) trypsin or pancreatic juice, and (4) blood. To do away with these sources 
of error they advise the rejection o' gastric contents showing any of these 
substances. Filtering the contents removes the peptid-splitting bacteria 
to some extent while the addition of toluol prevents their development. 
Tryptophan, as such, may be tested for as usual, while the presence of pan- 
creatic juice may be assumed if bile be present. Blood is shown by tests 
outlined under Feces. Warfield^ has recently shown another fallacy of this 
test, which may render the test of little clinical value. He has found that the 
saliva of many patients is capable of decomposing glycyl-tryptophan into its 
constituent elements. He concludes, therefore, that swallowed alkaline 
saliva, when mixed with neutral or faintly acid (not over 0.05 per cent. HCl) 
gastric contents, may introduce sufficient error to account for the discordant 
results obtained. Koelker^ has found a di- and tripepid-splitting ferment in 
the saliva. Lyle and Kober^ believe that a negative result with this test is of 
far greater value than is a positive one. 

Weinstein^ believes that the presence of tryptophan in the gastric con- 
tents, as withdrawn, is more positive as a test for carcinoma than is the glycyl- 

^ Ergebn. d. inn. Med., 1908, II, 212. 

2 Glycyl-tryptophan, as prepared for the test, is a clear solution. It is furnished by 
Kalle & Co., Biebrich a. Rhein, under the name of "Fermentdiagnostikum." It comes in 
small bottles which are used directly for the test by simply adding the 10 c.c. of filtered 
gastric contents and incubating for 24 hours. 

3 Bull. Johns Hopkins Hosp., 1911, XXII, 150. 

4 Ztschr. f. physiol. Chem., 1911, LXXVI, 27. 
•^ New York Med. Jour , 1910, XCI, 1151. 

* Jour. Am. Med. Assn., 1910, LV, 1085; Ibid., 191 1, LVII, 1420. 



GASTRIC CONTENTS 95 

tryptophan test, providing intestinal fluid has not regurgitated into the 
stomach. He has confirmed the findings of Warfield and asserts that this 
latter fallacy can not be applied to his "tryptophan test" as the salivary 
proteolytic action is not such as to produce tryptophan from protein in non- 
cancerous gastric contents. He, therefore, suggests the following modifica- 
tion. A portion of the gastric contents is withdrawn 4 to 5 hours after a full 
dinner and the filtered contents tested directly for tryptophan after acidifying 
the filtrate with acetic acid. If no reaction appears, incubate for 24 hours and 
repeat the test. Owing to the large number of possible fallacies and the 
discordant results obtained with this test, it seems probable that it, like so 
many others, must be regarded as of little diagnostic value. ^ 

Freidman and Hamburger (Arch. Int. Med., 1913, XII, 346) believe that 
the proteolytic cleavage of stomach contents is due, in most instances, to 
regurgitated trypsin, although leucocytes and bacteria play some role. They 
advocate a combined method of proteolysis and peptolysis, which they believe 
preferable to other tests advanced. A high peptolysis with low proteolysis 
speaks for carcinoma; while a high peptolysis and high proteolysis speaks 
against carcinoma. This test promises to be a reliable one but confirmatory 
work must establish its status. 

Wolff and Junghans' Test. 

These workers^ have originated a test for the differentiation of benign and 
malignant achylias, which test seems to have considerable diagnostic value if 
properly controlled and interpreted. The test is based on the following ob- 
servations. Normal aspirated test meals show a relatively large amount of 
soluble albumin, which arises from the digestion of the protein of the test meal 
by the gastric ferments in the presence of free hydrochloric acid. This albu- 
min increases in amount in proportion to the time which it remains in contact 
with the normal gastric juice. In conditions associated with achylia or with 
marked diminution of hydrochloric acid in the stomach, it would be reasonable 
to suppose that little or no protein digestion would occur. This was found to 
be the case in benign achylias but not true in the malignant types. In these 
latter cases the soluble albumin (probably a proteose) was found to be much 
increased. This albumin may arise (i) from protein retention from a pre- 
vious meal, due to stasis, lack of motor tone, or true obstruction, as shown by 
Clarke and Rehfuss; (2) from mixing of the test meal with so-called "cancer 
juice" exuded from the malignant growth; (3) from the presence of blood or 
pus arising from ulcerative lesions; (4) from the presence of swallowed saliva 

^ In this connection see Taylor and Hall, Jour. Path, and Bacteriol, 191 2, XVII, 121; 
Sanford and Rosenbloom, Arch. Int., Med., 191 2, IX, 445; Smithies, Ibid., 191 2, X, 357 and 
521; Jacque and Woodyatt, Ibid., 560; Hamburger, Jour. Am. Med. Assn., 191 2, LIX, 847; 
and Smithies, Ibid., 1913, LXI, 1793. 

2 Wolff and Junghans, Berl. klin. Wchnschr., 1911, XL VIII, 978; Wolff, Magen and 
Darmkrankheiten, BerUn, 1912, 217. See, also, Thiele, Berl. klin. Wchnschr., 1912, XLIX, 
544; Einstein, Med. Klin., 191 2, VIII, 484; Roubitschek and Weiser, Arch. f. Verdauungskr., 
1913, XIX (Erganz. Bd.), 146; Rolph, Med. Record, 1913, LXXXIII, 848; Smithies, Am. 
Jour. Med. Sc, 1914, CXLVII, 713; Trallero, Deutsch. med. Wchnschr., 1914, XL, 1438; 
Kahn and Jacobowitz, Biochem. Bull., 1915, IV, 214; Lenskaia, Russk. Vrach., 1915, XIV, 
267; Katznelson, Ibid., 292; Clarke and Rehfuss, Jour. Am. Med. Assn., 1915, LXIV, 1737. 
Friedenwald and Kieffer, Am. Jour. Med. Sc, 1916, CLII, 321. 



96 DIAGNOSTIC METHODS 

or, especially, sputum; and (5) from the action of a specific peptid-splitting 
ferment arising from the malignant growth and capable of splitting the pro- 
tein into lower groupings of aminoacids. This latter source is the most 
important, although the others must be excluded before proper interpretation 
is possible. In other words, one must be sure (i) that the stomach is free 
from remnants of previous protein meals, as in some cases this gastric re- 
siduum may be sufficient to give erroneous results;^ (2) that blood and pus are 
absent, at least in more than traces; and (3) that sputum especially is not 
swallowed during the period of retention of the test meal, as some types of 
sputum are relatively rich in soluble albumins. 

While, theoretically, typical cases of advanced gastric carcinoma, at least, 
should show little or no free hydrochloric acid, it should be remembered that 
such is actually not the case, so that this test applied to cases in which free 
hydrochloric acid is present to an appreciable extent may, of course, give 
high albumin values due to ordinary gastric digestion. However, the studies 
of Clarke and Rehfuss, by means of their fractional method of gastric exami- 
nation, show that the curve of protein digestion under normal conditions 
follows the acid curve rather sharply, while in malignant states the protein 
curve diverges from the acid curve and, as digestion progresses, this dispro- 
portion increases until there is a marked separation of the curves. From 
these facts, these workers argue that the fractional study of both the curves 
of gastric acidity and protein digestion should be simultaneously studied 
before the proper interpretation can be reached. 

Technic. 

In order to avoid the possibility of retention of protein residues in the 
stomach, the patient is allowed a light meal with very little protein on the 
evening before the examination is to be made. A light cathartic is adminis- 
tered the same evening. The next morning administer an Ewald test break- 
fast. Withdraw this meal within 45 to 60 minutes. Filter the contents 
through filter-paper until clear. 

While filtration is proceeding, arrange a series of six clean test-tubes in a 
rack, numbering the tubes serially. By means of a i c.c. pipet, which is grad- 
uated in divisions of i/ioo c.c, place in the respective tubes i c.c, 0.5 c.c, 
0.25 cc, 0.1 c.c, 0.05. cc, and 0.025 c.c. Now make up the volume in each 
tube to 10 cc. by the addition of distilled water from a finely graduated pipet. 
The dilutions in these tubes will be, then, i to 10, i to 20, i to 40, i to 100, i to 
200, and I to 400. Intermediate and extra dilutions may be made if it is 
desired to work the reaction down to a much finer point. Mix the contents 
of the tubes thoroughly by shaking and inverting the tubes. 

Overlay the contents of each tube with i cc. of the reagent to precipitate 
the albumin and note the appearance of a white ring at the point of contact of 
the fluid and reagent in the various tubes. A black background will bring 
out these rings more distinctly. 

Wolff's reagent is as follows: 

^ See Rehfuss, Bergeim and Hawk, Jour. Am. Med. Assn., 1914, LXIIT, 11. 






GASTRIC CONTENTS 97 

Phosphotungstic acid 0.3 gram. 

Concentrated Hydrochloric Acid i . o c.c. 

Alcohol (96 per cent.) 20.0 c.c. 

Distilled Water ad 200.0 c.c. 

Interpretation. 

Normally a distinct ring of albumin will be seen at dilutions of i to ic, i to 
20 and, occasionally, at i to 40. This reaction, which is called negative at 
these dilutions, may even reach, in very rare cases, as high as i to 200 due to 
various factors spoken of above, especially to the presence of blood and 
retention of protein residues. The importance of excluding extrinsic sources 
of albumin is thus evident. Generally speaking, however, the diagnosis of 
cancer becomes more probable the higher the dilution at which the albumin 
precipitation occurs, although cancer cannot be excluded in some cases when 
the precipitation does not appear in the last tubes. A precipitation unit of 
100 or over speaks rather markedly for a diagnosis of cancer, presumption be- 
coming almost a relative certainty when the unit is 200 to 400. This im- 
plies, of course, that all the points mentioned above hav-e been taken into 
consideration. Smithies regards a reaction at a dilution of 100 as doubtful. 
He states that this test " was a more constant finding in gastric extracts than 
were absent free hydrochloric acid, the presence of lactic acid and the gly- 
cyltryptophan test. It was rather more constant than tests for occult blood 
and the demonstration of motor inefficiency. It was not so consistent in its 
manifestation as the demonstration of organisms of the Boas-Oppler group 
or the increase in the formol index." 

It will be seen, therefore, that this test, of positive precipitation of albumin 
in dilutions of the gastric extract from i to 200 up, is of considerable value in 
the differentiation of the benign and malignant achylias or achlorhydrias, 
especially if the former are not associated with motor insufficiency. In cases 
of ulcer, especially if pyloric stenosis obtains, positive Wolff- Junghans' tests 
are not infrequent, indicating that symptomatology, gastric acidity and other 
findings must be taken into consideration. Like many other tests for cancer, 
this must not be relied upon to settle the diagnosis, to the exclusion of other 
laboratory and clinical data. With proper technic and care in excluding 
extrinsic factors, this test has certainly more than a presumptive value, giving 
definitely positive findings in fully 80 per cent, of disputed cases. 

BIBLIOGRAPHY 

1. Boas. Die Erkrankungen des Magens. Berlin, 1906. 

2. Billings. Diseases of the Digestive System. New York, 1906. 

3. Debove, Achard, et Ca'staigne. Manuel des Maladies du Tube Digestif. 

Paris, 1907. 

4. EwALD. Die Erkrankungen des Magens. Berlin, 1900. 

5. Harley AND GooDBODY. The Chemical Investigation of Gastric and Intestinal 

Diseases. London, 1906. 

6. Hemmeter. Diseases of the Stomach. Philadelphia, 1906. 

7. LoEPER. Manuel des Maladies du Tube Digestif. Paris, 1907. 



98 DIAGNOSTIC METHODS 

8. Lyon. Diagnostic et Traitement des Maladies de L'Estomac. Paris, 1909. 

9. Reed. Diseases of the Stomach and Intestines. New York, 1907. 

10. RiEGEL. Die Erkrankungen des Magens. Wien, 1903. 

11. RoESER. La Chimie Alimentaire. Paris, 1906. 

12. SiGAUD. Traite clinique de la digestion. Paris, 1907. 

13. Smithies and Ochsner. Gastric Cancer. Philadelphia, 1915. 

14. Stockton. Diseases of the Stomach and Their Relation to Other Diseases. 

New York, 19 14. 

15. ZuNz. Methodik der Mageninhaltuntersuchung, Handb. d. Biochem. Arbeits- 

Methoden, 19 14, VIII, 44. 

16. ZwEiG. Diagnose und Therapie der Magen- und Darmkrankheiten. Wien, 

1913- 



CHAPTER IV 
THE FECES 

I. General Considerations 

The feces are composed of substances of different origin, which may be 
'divided as follows: (i) Food remnants, either undigestible constituents or 
digestible but unabsorbed elements; (2) secretions of the alimentary tract; 
(3) decomposition products and bacteria; (4) formed and unformed elements 
derived from the intestinal wall, and (5) foreign bodies, such as hair, wood 
fiber, parasites, parasitic ova, and enteroliths. It is not possible to draw a 
, sharp line between a truly physiologic and pathologic composition of the feces. 
!ln each individual case this division will depend upon a number of factors, 
among which may be mentioned composition of the food, method of taking 
the food, individual functional capacity of the intestines, frequency of the 
^bowel movements, and general systemic conditions. The condition of the 
food remnants will give much information regarding the functional capacity of 
the bowel. Under normal conditions, about one-third of the dry substance 
of the feces arises from the bacteria, this amount increasing under certain 
pathologic conditions which will be later discussed. As products of bacterial 
activity we observe the formation of hydrobilirubin from bilirubin as well as 
the reduction of certain medicaments and the rare formation of certain 
diamins, which will be treated later. This bacterial activity will vary much 
,under pathologic conditions and may have much to do with the symptoma- 
[tology of the case investigated. ^ The ordinary decomposition products are 
"derived from the carbohydrates and proteins of the food; from the former are 
produced, by fermentation, volatile fatty acids, lactic acid, succinic acid, 
alcohol, carbon-dioxid, hydrogen, and methane, while from the latter are 
[formed by putrefaction indol, skatol, phenol, ammonia, and hydrogen sul- 
i'phid. The fats are decomposed only to a very slight extent. The normal 
products of digestion in the intestinal tract as well as the factors bringing 
about these changes will be discussed later. ^ 

Normal Feces. 

For proper comparisons between the feces of various individuals the food 
must be the same. Starvation feces, meat feces, and milk feces are typical 
types, but are not normal in the strict sense. For comparison, however, an 
arbitrary norm must be established in order to judge of slight variations which 
have to do with special differences in utilization of food and which are not 
observable by the eye except under certain conditions (as, for instance, fatty 

^ See von Noorden, Jour. Am. Med. Assn., 1913, LX, loi. 

^ See Sanford, Wisconsin Med. Jour., 1914, XII, 281; also, McClanahan and Moore, Am. 
Jour. Med. Sc.,1915, CXI.IX, 815; Bergeim, Jour. Biol. Chem., 1917, XXXII, 17. 

99 



100 



DIAGNOSTIC METHODS 



Stools). Through the work of Praussnitz and of Schmidt and Strasburger a 
new field for study of normal and abnormal feces has been opened up. 

A normal feces should be one which consists almost entirely of remnants 
of the digestive juices and intestinal secretion arising from a purely digestible, 
properly prepared, and assimilable food. It contains approximately 8.6 per 
cent, of nitrogen, i6 per cent, of ether extract, and 15 per cent, of ash calcu- 
lated on the dry basis. Any appreciable variation from this composition 
would indicate a diminution of the functional activity of the bowel. In the 
investigation of pathological cases this composition is scarcely to be assumed, 
as the diet is more or less restricted, the appetite is capricious, the intestinal 
activity is variable, and the feces are, therefore, different from those of the 
healthy. It must not be understood from this that a feces showing the 




Fig. 19. — Normal Feces. (JLandois.) 
a, Muscle fibers; h, tendon; c, epithelial cells; d, leucocytes, e-i, various forms of plant- 
cells, among which are large numbers of bacteria; between h and h are yeast-cells; k, 
ammonium-magnesium phosphate. 

above composition is necessarily normal in other respects. One may not 
rely entirely on the chemical composition, but must largely consider the 
macroscopic and microscopic findings. In order that we may have a sound 
basis upon which to judge of intestinal activity it seems wise to have some 
sort of test diet which may be given to suspected cases. 

Diet of Schmidt and Strasburger. 

This diet^ is so selected that it can be used by the healthy as well as by 
those with intestinal trouble; its amount is sufficient to satisfy the maximal 
calorie requirement of the individual while at rest; it contains the three chief 
groups of food material in definite relation to each other; is as free as possible 
from remnant-leaving food, and can be easily obtained and prepared. The 
starchy food present is of the amount and kind which have been shown most 
favorable for the prevention of excessive fermentation in the bowel. The 
daily diet is as follows: One and five-tenths liters of milk, 100 grams of 
zwieback, two eggs, 50 grams of butter, 125 grams of beef (raw weight), 190 
grams cooked potato, and a gruel of 80 grams of oatmeal. This is distributed 

^ Die Faeces des Menschen, Berlin, 1915. 



► 



THE FECE^ lOI 

through the day as may best suit the patient. This diet contains 102 grams 
of protein, iii grams of fat, and 191 grams of carbohydrate, yielding 2,234 
raw calories. In cases which show a diarrhea, due to the milk, one may sub- 
stitute instead of J^ Hter of milk the same amount of cocoa made from 20 
grams of cocoa powder, 10 grams of sugar, 100 grams of milk, and 400 grams 
of water. Small variations in the amount of milk, sugar, butter, and even of 
eggs may be permitted, but the outline, as regards meat, zwieback, potato, 
and gruel should be rigidly adhered to. 

This diet should be administered for three days or longer if necessary to 
obtain a stool which comes from it. In order to judge of the first appearance 
of the stool from this diet, the patient should be given a capsule containing 0.3 
gram of powdered carmin, both preceding and following the diet.^ Instead 
of carmin, one may use cork, charcoal, or silicic acid. 

Folin's Diet. 

This diet^ is especially serviceable in case one wishes to follow the metabo- 
Hsm in any special case. Its easy application and its fairly constant values for 
nitrogen, phosphorus, chlorin, and sulphur make it invaluable. 

The standard diet, which is given to the patient daily for several days, is 
as follows: 



Whole milk. 






500 


c.c. 


Cream (18 to 22 


per cent. 


fat). 


300 


c.c. 


Eggs (whole), 






450 gm. 


Horlick's malted milk, 




200 


gm. 


Sugar, 






20 


gm. 


Sodium chlorid. 






6 


gm. 


Water, 






2,100 


c.c. 



This diet contains approximately 119 gm. of protein, 148 gm. of fat, and 
225 gm. of carbohydrate, yielding 2787 raw calories. The intake of one day is 
nitrogen 18.9 gm., 5.9 gm. of P2O5, 3.8 gm. of SO3, and 6.2 gm. of CI. It is 
not to be compared with that of Schmidt and Strasburger for estimating the 
intestinal activity, but is more reHable if metaboHsm relations are to be 
studied. 

The normal motility of the feces is from 6 to 20 hours, while on a milk diet 
it may vary from 36 to 48 hours. This factor may be of importance in judg- 
ing of intestinal obstruction. The isolation of a stool under a special diet is 
of great importance, as the success of an investigation will depend upon this. 

Obtaining Intestinal Juice. 

Boas has shown that it is often possible, by the use of the stomach tube, 
to obtain sufficient intestinal secretion to permit of examination. His technic 
is, however, rather difficult, is trying to the patient and is often unsatisfactory. 
Boldyreff, Volhard, Faubel and, more recently, Koziczkowsky^ have shown 

1 See Basch, Jour. Am. Med. Assn., 1913, LXI, 1295; Strauss, Arch. f. Verdauungskr.,. 
1914, XX, 299; Hymanson, Am. Jour. Dis. Child., 1916, XI, 112. 

2 Jour. Physiol, 1905, XIII, 45. 

3 Ztschr. f. klin. Med., 1909, LXVIII, 261. 



I02 DIAGNOSTIC METHODS 

that the intestinal juice may easily be obtained by the following method. The 
patient is allowed to take 150 c.c. of pure olive oil when the stomach is empty, 
preferably in the morning. At the end of three-fourth hour, during which 
time the patient must remain quiet in a sitting posture, the gastric contents 
are removed by means of a stomach tube. The material, obtained by means 
of this oil test-breakfast,^ readily separates into two layers, an upper of oil 
and a lower of the mixed gastric and intestinal secretions. 

Trypsin is best detected by the method of Gross. ^ Either the fluid ob- 
tained as above or a portion of the feces, rubbed up with three times its amount 
of a I to 1 ,000 Na2C03 solution and filtered, may be used for the test. 100 c.c. 
of a 0.5 per thousand solution of casein in i to 1,000 Na2C03 solution are 
treated with 10 c.c. of fluid mentioned above and the mixture placed in the in- 
cubator for eight to twelve hours. At the end of this period the addition of a 
few drops of a dilute (i per cent.) acetic acid should produce no precipitate in 
cases digestion is complete, that is if trypsin is present. 

Functions of the Intestinal Juices. 

The pancreatic juice as excreted into the intestine is an alkahne fluid^ con- 
taining three ferments, trypsin which hydrolyzes protein, amylopsin which 
acts upon the carbohydrates, and lipase (steapsin) which aids in the digestion 
of fat. The trypsin appears to be excreted in the form of a zymogen which 
is activated by a second ferment, enterokinase, derived from the intestinal 
mucosa, while the lipase and amylopsin are active when secreted. It has been 
shown by Pawlow that the passage of free hydrochloric acid into the duode- 
num is a direct stimulant to the excretion of pancreatic juice. Bayhss and 
StarHng believe, however, that the stimulus to the pancreatic secretion is not 
the free acid but is a ferment, secretin,^ which is formed by the action of the 
hydrochloric acid upon the intestinal mucosa. While the trypsin acts upon 
protein bodies, splitting them through various stages into the ultimate prod- 
ucts, amino-acids and hexone bases, there is a second ferment, erepsin, 
discovered in the intestinal mucosa by Cohnheim, which acts upon the 
intermediate splitting products of protein, such as the albumoses and pep ton, 
carrying this conversion to the same lengths as does trypsin.^ It is inter- 
esting to note that we find in the intestinal juice of the infant a ferment, 
lactase, which hydrolyzes lactose into the simpler saccharids. 

The presence of the bile, which reaches the duodenum through the 
common duct, is necessary for the proper digestion and absorption of the 
fatty substances of the food. Variations in this constituent may reflexly 
cause disorders in the secretion of the stomach, one finding very frequently 

^ See Matko, Med. Klin., 1913, IX, 1546 and 1587. 

2 Deutsch. med. Wchnschr., 1909, XXXV, 706. See Izar, Policlinico, 1916, XXIII, 
741. 

3 Long and Fenger, Jour. Am. Chem. Soc, 1915, XXXVII, 2213; Ibid., 1916, XXXVIII, 
1 1 14; Long, Hull and Atkinson, Ibid., 1915, XXXVII, 2427; Long and Hull, Ibid., 1916, 
XXXVIII, 1620; Ibid., 1917, XXXIX, 162; Ibid., 1051; Fenger and Hull, Jour. Biol. 
Chem., 1919, XXXVIII, 487; Wago, Arch. Int. Med., 1919, XXIII, ZZ and 251. 

^ See Carlson, Lebensohn and Pearlman, Jour. A. M. A., 1916, LXVI, 178. 

^ See Schlecht and Wittmund, Deutsch. arch. f. klin. Med., 1912, CVI, 517; Friedmann, 
Med. Record, 191 2, LXXXI, 355; Abderhalden,Ztschr.f. physiol. Chem.,1912, LXXVIII, 
344. 



THE FECES IO3 

hyperacidity associated with obstructive jaundice. Whether the bile may be 
assumed to have a disinfecting power must be left for the future. 

Estimation of Intestinal Digestion. 

A careful chemical examination of the feces, coupled with a macroscopic 
and microscopic investigation, will give much information regarding the 
degree of intestinal digestion and absorption. However, such methods are 
time-consuming and not easily performed by the general practitioner. For 
this reason Sahli has introduced a method, similar to his stomach method, of 
investigating such activity. He employs glutoid capsules, which are made 
from gelatin hardened with formaldehyd. These capsules either do not 
dissolve in the gastric juice at all or only after considerable time, although 
they are quickly soluble in the intestinal juice. In these capsules is placed 
material which will not diffuse through the capsule wall and whose absorption 
may be studied from an examination of the saliva or urine. Sahli uses either 
iodoform or salol. In the former case o. 15 gram of iodoform is placed in a 
glutoid capsule, and given with an Ewald test meal. Under the best condi- 
tions (normal gastric motility, normal intestinal digestion, and normal intes- 
tinal absorption) the iodin reaction may be expected to appear, according to 
Sahli, in the saliva within four to six hours; that is, within one and one-fourth 
hours after the capsule has been dissolved by the pancreatic juice. Instead 
of iodoform salol may be used, being given in the amount of one-half gram 
along with an Ewald test meal. The reaction for salicyluric acid may be 
obtained in the urine within one and one-half hours after the capsule has been 
taken. 

Sahli gives the following results obtained by the use of this capsule: When 
the stomach contains neither free hydrochloric acid nor pepsin, the reaction is 
not delayed so long as gastric motility is good. In cases of diarrhea due only 
to an increased peristalsis without any marked disturbance of intestinal diges- 
tion, the reaction is either normal or even somewhat hastened. In other 
types of diarrhea characterized by an involvement of the intestinal chemistry 
or intestinal absorption, the reaction is either absent or distinctly delayed or 
the capsules may be found undigested in the feces. This method also aids in 
differentiating an icterus due to occlusion of the ductus choledochus at its 
point of entrance into the intestine, in which case the digestion of the capsule 
may be interfered with, from one where the obstruction to the bile flow is 
higher up near the liver. This test may be of some presumptive evidence in 
the diagnosis of pancreatic carcinoma, although all cases of pancreatic car- 
cinoma do not necessarily occlude the duct, in which case a positive result 
would obtain. Galli reports a case of carcinoma of the pancreas in which the 
glutoid capsule was dissolved, although no pancreatic juice was present in the 
bowel. 

II. Macroscopic Examination 
(i) Method. 

The macroscopic examination of the feces embraces not only the study of 
the physical characteristics, but also a recognition of various normal and 
abnormal substances. It is convenient, when examining the feces, to employ 



104 



DIAGNOSTIC METHODS 



some form of a washing apparatus to separate the coarser from the finer 

particles. 

Boas has introduced a special sieve for such work which, however, does not 

have much advantage over the ordinary flour-sifter which Einhorn advises. 

Strauss' method of washing the feces by a 
current coming from below seems to be much 
the best of any of these types of apparatus. 
In the absence of any other equipment, an 
ordinary household sieve of various-sized 
mesh will answer the purposes. Small 
amounts of mucus or connective tissue frag- 
ments may be recognized by rubbing up a 
portion of the stool in a mortar with a little 
water, when these substances will float upon 
the surface. In some cases it may even be 
advisable to place the feces in tall glass jars 
in which the stool is mixed with water and 
allowed to arrange itself in layers. 

If the stool is of ordinary consistency, it 
should be spread out in a more or less thin 
layer so that the larger particles may be 
easily recognized. If it be, however, a 
watefy stool the contents are thoroughly 
mixed and examined as such. 




Fig. 20. — Boas' stool-sieve. 

{Hemmeter.J 



(2) Amount. 

The amount of feces excreted will de- 
pend upon (i) the quantity and quality of the food; (2) the remnants of 
intestinal juices and debris; (3) the condition of the digestive organs, and (4) 
the bacteria. These factors are all important, each being dependent to a 
certain extent upon the other. 

The average moist weight may be considered as varying from 100 to 250 
grams with a dry weight of 20 to 40 grams. This amount may, however, 
reach as high as 20 kilograms as in a case reported by Lynch. 

Number of Stools. 

The number of stools which may be passed in 24 hours is subject to very 
wide variation, even under physiological conditions, but is usually constant 
in the same individual. At least one stool a day should be considered normal, 
although many persons are accustomed to have only one movement of the 
bowels in 48 hours and others in longer periods. We must, therefore, consider 
each individual case before judging as to abnormality in the number of stools. 
Lynch^ has reported a case of a patient having only one stool each 100 days, 
while Geib and Jones^ discuss a case in which there was no stool during an 
entire year the patient at the end of that time voiding 32 liters of feces. 

A diarrhea is said to exist when the stools are frequent and fluid. The 

1 Thesis, Buenos Aires, 1896. 

2 Jour. Am. Med. Assn., 1902, XXXVIII, 1304. 



THE FECES I05 

frequency may vary from 2 to 50 in 24 hours, although a single liquid stool in 
24 hours may constitute a diarrhea in a patient unaccustomed to having a 
movement every day. A normal stool is never fluid, so that the character 
of the stool is of more importance than the number, as individual peculiarities 
must be considered. A diarrhea may be due to increased peristalsis, to in- 
creased intestinal secretion, a diminished gastric secretion, or a decreased 
absorption from the bowel. The most extreme grades of diarrhea are ob- 
served in Asiatic cholera, dysentery and the summer diarrhea of infants, al- 
though a marked diarrhea does occur in enteritis, peritonitis, intestinal 
tuberculosis, and uremia. Some infectious diseases are more frequently 
associated with diarrhea than are others, but little of diagnostic importance 
is obtained from this symptom. 

By constipation is meant the infrequent and irregular movement of the 
bowels, associated with symptoms which are relieved by administration of 
laxatives. The habits of the individual must be taken into consideration 
before judging as to a real or apparent constipation. This condition is physio- 
logically a result of a sedentary life or of a diet lacking in elements which will 
stimulate intestinal peristalsis.^ Pathologically, we find constipation in cases 
of dilated stomach, occulsion of the bowel, atony of the bowel wall, in con- 
ditions causing increased cerebral pressure, and in obstruction by pressure 
from without the bowel. It must be stated that we may have an apparent 
diarrhea as a symptom of constipation high up in the bowel. It is better 
practice to administer a laxative in such conditions than it is to give an 
astringent. 

(3) Consistency and Form. 

The consistency and form of the normal stool vary considerably depending 
upon the nature of the food ingested. The stool is much softer with a purely 
vegetable diet, of which about 85 per cent, is water, than with an animal diet, 
of which only 65 per cent, is water. One differentiates, as regards consistency, 
a well-formed, a mushy, and a fluid stool, between which types there are many 
gradations, some stools being partly formed and partly fluid. Many factors 
may influence the amount of water present in the stool, such as (i) a lessened 
absorption of water from the intestinal canal; (2) intake of a large amount 
of water, and (3) an increased secretion from the intestinal glands. 

The consistency of the normal stool varies from day to day and can be 
constant only when the patient is placed upon a standard diet. It may be 
abnormally too fluid or too solid, in the latter case being frequently voided in 
the form of very hard masses, known as " scybala." As a general rule, it may 
be stated that the greater the absorption of water from the intestine the more 
firm will the feces be and, in consequence, the more frequently will these scybala 
form. Stools are frequently observed of which the consistency is normal 
but of wh'.zh the size of the cylinder is quite small. Such small "lead- 
pencil" sized stools have been supposed to be indicative of stricture of the 
lower bowel, but this condition is not necessarily present. 

1 See Cannon, Jour. Am. Med. Assn., 191 2, LIX, i. See Mellanby, Quart. Jour. Med., 
1916, IX, 165. 



I06 DIAGNOSTIC METHODS 

Besides the water-content of the feces, the amount of fat, mucus, and 
vegetable residue has much to do with the consistency of the stool. One may 
differentiate the fat from water in the stool by placing a small portion of the 
feces upon a shde and pressing a cover-slip down upon it. If the cover-glass 
remains when the pressure is removed increased fat may be assumed, while 
if the softness of the feces be due to increased water the cover-glass will 
spring away from the feces. 

In some cases, especially those associated with achylia gastrica, the stools 
are very frothy, indicating a marked bacterial decomposition. Such stools 
should not be confused with those of the ordinary diarrhea or with the char- 
acteristic "rice-water" stools of Asiatic cholera, in which particles of mucus 
are readily detected. Such stools are never found associated with large fat 
contents. 

(4) Odor. 

The peculiar odor of the normal stool is referable to the presence of indol 
and skatol, which arise from the putrefaction of protein material in the large 
intestine. Along with these we may find other substances, such as hydrogen 
sulphid, methane, and phosphine. The odor of the stool is much more 
marked following a meat diet than that after a vegetable diet; it is very slight 
on a milk diet and is practically lost in the fasting condition. If the processes 
in the intestine are of such a nature that fermentation of the carbohydrates 
exceeds the putrefaction of the proteins, the stool shows a distinct sour odor 
traceable to the presence of butyric or acetic acid. The odor in cases of acute 
and chronic diarrhea is frequently very slight, while that of the loose, watery 
discharges of cholera is peculiar and sperm-like, referable to the presence of 
cadaverin. In the diarrhea of children a distinct putrid odor may be present, 
although this is not necessarily the case. The so-called acholic stools have in 
themselves, according to Schmidt, very little odor, showing this property only 
when complicated by decomposition processes arising from the lack of bile. 
In cases of severe dysentery and carcinoma of the large intestine an intensely 
disagreeable odor is observed which differentiates these conditions from 
those associated with other types of decomposition. 

(5) Color. 

The color of the stool varies from a light brown to even a black, depending 
upon the kind of food, the residue of the intestinal secretions, the presence of 
pathologic products from the intestinal wall, and the administration of thera- 
peutic agents. The dark color of the normal stool is due to the presence of 
hydrobilirubin, which is formed from the secreted bilirubin by the reducing 
processes in the intestines. Bilirubin is found normally in the stool of a 
nursing child, being converted either into biliverdin or hydrobilirubin under 
abnormal conditions. This change from a light yellow infantile stool to a 
greenish one has much importance to the pediatrician. A well-formed stool 
is always darker in color than is the thin stool, which is equivalent to saying 
that the stool is darker the longer it remains in the intestine. The color of a 
stool under a meat diet is dark brown, with a vegetable diet a lighter brown, 
and following a milk diet a fight yellow. This color which is traceable to the 



I 



THE FECES 107 

diet is shown best in those cases in which bile is excluded from the bowel. The 
dark color of the meat feces is probably traceable to the conversion of the blood- 
coloring matter into hematin and not to the formation of sulphid of iron which is 
so commonly stated. Food products may color the feces a characteristic shade. 
Thus, coffee may give a dark brown color, cocoa a brownish-red color, red 
wine a smoky black-brown color which has a shade of green. Chloropyll- 
containing plants, such as spinach and lettuce, give rise to greenish shades. 

Occasionally the reduction process in the intestines may go as far as to 
convert the bilirubin into leucohydrobilirubin instead of into hydrobilirubin. 
This hydrobilirubin is identical with the urobilin found in the urine. Such 
stools may be practically colorless when voided, but will be converted into the 
dark brown normal-colored stool on contact with the air. Besides these pig- 
ments, biliprasin has been isolated from the feces by Fleischer, while Miiller 
obtained cholecyanin. 

The variations in color of the stool following administration of therapeutic 
agents is frequently characteristic. Thus, after the use of calomel one ob- 
serves a distinct green coloration due to the conversion of the bilirubin into 
biliverdin. Bismuth preparations color the stool a distinct black, due to the 
formation of the oxid or sulphid of bismuth. Rhubard, senna, santonin, 
and gamboge cause a distinctly yellow coloration which will change to a 
reddish tone in the presence of alkali. Iron compounds produce shades rang- 
ing from dark brown to black while methylene blue gives rise to the produc- 
tion of a blue-green color and sandal- wood a reddish- violet color. 
Pathologic Variations in Color. 

Cases showing the presence of large amounts of -mucus or of pus in the 
feces are characterized by a gray-white or yellowish-gray coloration of the 
feces. Such cases are seen in membranous colitis and rupture of an abscess, 
especially of the appendiceal variety, into the intestinal tract. In cases of 
syphilitic or carcinomatous ulceration of the colon or rectum this character- 
istic color tone may be more or less influenced by the presence of blood. 

Stools showing the presence of a large amount of fat are clay colored. 
That this coloration is due to the excess of fat, rather than to the absence of 
bile, may be shown by extracting the feces with alcohol and ether, in which 
extraction the bile will be taken up along with the fat and will color the sol- 
vents. These acholic stools as they have been called occur both in cases 
associated with biliary obstruction and in those showing no obstruction. 
It would be better practice, therefore, to style these stools colorless, instead of 
acholic stools. Striimpell was able to obtain stools of a light brown color by 
feeding patients a diet containing small amounts of fat, thus proving that the 
increased fat was more important than the dimunition of bile, although this 
latter does account for some cases. This clay-colored stool may also be 
found in diarrhea, while in Asiatic cholera and dysentery the stools may be 
absolutely colorless. They have been also found in cases of leukemia, carci- 
noma of the stomach or intestine, tuberculous enteritis, and chronic tuber- 
culous peritonitis. The cause of this lack of color may be the same unknown 
cause that produces the formation of leucohydrobilirubin spoken of above. 



I08 DIAGNOSTIC METHODS 

In some cases we find a distinct golden-yellow or even a green color of the 
feces. This is due to the presence of unaltered bile, on the one hand, and bili- 
verdin, on the other, Biliverdin is usually found in abnormal decomposition 
processes in the intestine of the infant, while unaltered bile may appear in 
cases of increased intestinal peristalsis, in which the contents of the bowel are 
rushed onward before absorption can take place. This is probably the 
partial explanation of the green stool following the administration of calomel. 
Normally, bilirubin is not found in the intestine below the ascending colon. 
Such being the case, it may be possible to judge of the point of irritation in a 
diarrheal attack by the fact that the higher up in the bowel the point of dis- 
turbance the more of this pigment will be found in the feces. Bilirubin in- 
dicates, therefore, an enteritis especially of the small, but also of the large 
intestine. This bile pigment may be found most frequently on cellulose 
material, mucus, muscle fibers, and fat. It is readily detected by rubbing up 
2 or 3 c.c. of the feces with a concentrated aqueous solution of pure mercuric 
chlorid. This mixture is allowed to stand 24 hours and is examined micro- 
scopically thereafter. The fragments to which the pigment is attached wiU 
stain red if due to hydrobilirubin, while those stained with bilirubin will 
show a green color. Naturally, in this examination chlorophyll-containing 
fragments must be excluded. The most favorable material for such exami- 
nation is the mucus. A green color of the stools may also be observed due 
to infection with the bacillus pyocyaneus. 

Blood may give rise either to a distinct red color, a brownish-red shade, 
or even a black tint. If the blood be adherent to the scybalous masses or to 
the well-formed feces, it is usually derived from the rectum or anus and indi- 
cates hemorrhoids; if it be evenly distributed with the food material and is 
changed from a bright red to a brownish color, it indicates a hemorrhage in 
the stomach or high up in the small intestine, especially if the stool be solid; 
while an evenly mixed bloody feces of fluid character will usually point to the 
colon as the seat of the trouble. As a general rule, it may be said that the 
darker the color the more remote from the rectum will be the hemorrhage. 
Tarry black blood is seldom of low origin, usually indicating trouble in the 
stomach or duodenum, while fluid scarlet blood usually arises from the colon 
or rectum, although in some cases of typhoid fever the blood may be a bright 
red, although the hemorrhage may be fairly well up in the bowel. In cases 
of intussusception the blood may appear mixed with serum, but with no 
fecal matter. 

In deciding as to the importance of blood in the feces one must naturally 
exclude that arising from food or from hemorrhages above the gastro-intes- 
tinal tract. Thus blood coming from the mouth, nose, throat, or lungs may 
pass into the stomach and out with the feces, making a mistake in diagnosis 
very possible. Further, blood arising from vaginal discharges, which may be 
mixed with the feces at the time of defecation, should be excluded. 

The detection of blood in the feces is more or less simple and direct in the 
fresh state, but when intimately mixed with the feces its recognition is a 
matter of some difficulty. In many cases of hidden or occult bleeding blood 



r 



THE FECES lOQ 

is never detected microscopically or macroscopically. It is practically useless 
to search for blood-cells in the feces, as rarely are perfect cells found, unless 
the blood is present in very large amount, many specimens showing no cells. 
Guaiac Test (Van Deen^s Test). 

A small portion of the stool is rubbed up with water and one-third of its 
volume of glacial acetic acid added. This mixture is well shaken in a test- 
tube and a few cubic centimeters of ether added. After thoroughly shaking 
this mixture, it is allowed to settle, when the ether, in the presence of blood, 
will have assumed a brownish color. In case the ethereal extract is not clear, 
a few drops of alcohol may be added. On adding to this ethereal extract a 
mixture consisting of equal parts of fresh tincture of guaiac' and ozonized 
turpentine, a blue ring will form at the point of contact or a blue coloration 
wiU be seen throughout the mixture if the tube be shaken. 

This test is much more rehable in its negative phase than in its positive 
phase. The writer has frequently found positive tests for blood arising from 
the employment of tubes previously used with copper solutions or with nitric 
acid, so that he would advise the worker invariably to use either new or 
thoroughly clean test-tubes when testing for the presence of blood. More- 
over, this test is given by other substances, which may react positively. If 
the patient eats watermelon, prunes, potatoes or rice, has been taking iron, or 
the feces contain much pus, a distinct reaction may be present.- In this test 
as well as in all of the other tests outhned, the presence of hematin arising 
from the meat of the diet must be excluded. This can be done only by placing 
the patient upon an absolutely meat-free diet for several days preceding the 
examination. 
The Schaer-Kltmge Test. 

This test is very similar to the preceding, but is much more delicate, being 
positive after the ingestion of only three grams of blood. It is even more im- 
portant when using this test to exclude all hemoglobin and chlorophyll- 
containing foods for some days preceding the examination. The stool is 
rubbed up with water and treated as in the preceding test with acetic acid and 
ether. To this ethereal extract is then added a mixture of i c.c. of ozonized 
turpentine and 3^ c.c. of fresh 3 per cent, alcoholic aloin solution. This may 
be prepared by dissolving what aloin will he on the point of a spatula in 3^ 
of a test-tube of 95 per cent, alcohol. At the line of contact a distinct red ring 
will be observed in the presence of blood in from three to five minutes. Care- 
ful work with this test has shown that fat interferes to some extent with its 
deHcacy. It is, therefore, customary to treat the feces with an equal volume 
of ether and to shake thoroughly to remove all the fat present. The ethereal 
solution is then poured off and the remaining fecal material mixed with one- 
third its volume of glacial acetic acid and 10 c.c. of ether, being then 
thoroughly shaken and set aside. 

^ ^Lyle and Curtman, Jour. Biol. Chem., 1918, XXXIII, i, advise the use of guaiaconic 
acid. See Boas, Beil. klin. Wchnschr., 1916, LIII, 1357. 

2 BoUing the watery suspension of the feces and allowing it to cool before adding the acid 
and ether wiU prevent reactions arising from ferments and oxygen transmitters other than 
blood; Rapin (Rev. Med. de la Suisse Rom., 1916, XXXVI, 235) shows that charcoal, given 
to mark the feces, may act as a catalyser in various tests for occult blood. 



no DIAGNOSTIC METHODS 

A portion of this brownish ethereal extract, which contains the hematin 
formed by the action of the glacial acetic acid upon the hemoglobin of the 
blood, is poured into a thoroughly clean test-tube and treated as above de- 
scribed with the turpentine and aloin solution. If the tube be shaken after the 
red contact ring has formed the whole mixture will assume a cherry-red color. 
If the tube is allowed to stand for a few minutes the aloin solution may sink to 
the bottom, forming a distinct red layer beneath that of the ether and turpen- 
tine. A reaction to be positive should appear within lo minutes as the aloin 
itself will gradually turn red under the conditions of the experiment if left for 
a much longer period. Charcoal instead of carmin should be used to mark 
the feces. The writer has found this test very reliable and very easy of 
application. 

Weber's Test. 

This test has the advantage of excluding practically every other factor 
which might influence the tests previously given. ^ A portion of the feces is 
extracted with ether to remove the fat and is then separated from the ethereal 
solution. This fat-free feces is then rubbed up with water and treated with 
glacial acetic acid and ether. The ether, as in the preceding test, takes up 
the hematin which is now detected by the spectroscope, showing the char- 
acteristic spectrum, namely, an intense narrow band in the red between C and 
D and a somewhat more definitely marked group of three broader bands, (i) 
in the yellow, (2) at the boundary between yellow and green, and (3) at 
the boundary between the green and blue, this last band being difl&cultly 
recognizable. 

In order to avoid confusion with the spectrum of methemoglobin or of 
chlorophyll, one may convert the hematin into reduced hematin (hemochro- 
mogen) by the addition of alcohohc potassium hydrate, water, and ammonium 
sulphid solution. The spectrum of this latter substance is characterized by 
the two bands in the green. 

This test is probably the most sensitive and should be more frequently 
employed. It is very simple of application, is very reliable and has practi- 
cally no fallacy, especially when the hematin is converted into the hemo- 
chromogen. This test is, however, not so dehcate as the aloin test. 

Weber originally used the guaiac test with a modification of extracting 
the fat from the feces before testing with guaiac and turpentine. Schumm 
states that if the feces be thoroughly extracted with alcohol and ether most 
of the fat and urobilin will be removed and that under these circumstances 
Weber's test, either with the guaiac tincture and turpentine or with the spec- 
troscope, becomes much more valuable. 
Adler's Test. 

O. and R. Adler have introduced the use of benzidin as a test for the 

presence of blood. The stool is extracted with a mixture of alcohol and ether 

for the reasons above mentioned. It is then treated with glacial acetic acid 

and with ether as described in the other tests. This acid ethereal extract, 

^ Queisser (Therap. Monatsh,, 1913, XXVII, 727) found blood by this test in cases fed 
upon the Lenhartz gastric ulcer diet, such cases being free from gastro-intestinal ulceration. 



THE FECES III 

which contains the hematin, is then treated with 2 c.c. of a saturated alcohohc 
benzidin solution and 2 c.c. of hydrogen peroxid (3 per cent.). In the pres- 
ence of blood a greenish-blue color appears. 

Wagner^ advocates a so-called "dry test" of the stool which is delicate, 
reliable and extremely simple. He uses as reagent the Schlesinger-Holst 
mixture of a knife-point of benzidin, 2 c.c. of glacial acetic acid and 20 drops 
of a 3 per cent, solution of hydrogen peroxid. If a few drops of this mixture 
be poured over a little of the sohd feces spread on a clean glass shde, a greenish- 
blue color will appear almost immediately in the presence of blood. This 
benzidin test is very sensitive and can be recommended for general use. 

(6) Mucus. 

'^ From the diagnostic standpoint the recognition of much mucus in the 
feces is of the greatest importance. Any amount of visible mucus should be 
considered pathological, although it is to be remembered that mucus may be 
increased physiologically as the result of hypersecretion, in which case it will 
appear as a slimy coating of the scybalous masses or as small adherent par- 
ticles. Boas regards the mucus found after strong cathartics as normal, but 
this is to be questioned as the irritation may be sufficient to set up a mild hyper- 
secretion. The mucus expelled with the meconium and, according to Lynch, 
even that passed by infants up to the second week of life should be considered 
normal. The fecal mucus is a true mucin, being precipitated by acetic acid, 
but dissolving in 10 per cent. HCl. 

The larger portions of mucus maybe easily recognized with the naked eye, 
but the smaller bits are more clearly brought out by rubbing up the feces with 
water and holding the material, in a thin layer, toward the light. If the feces 
be well formed, the mucus may be separated from the exterior of the cylinder, 
as it is never found in the interior of a firm feces. In mushy stools the mucus 
is intimately mixed with the fecal material, usually in the form of smaller 
particles, the exterior type of mucus being of much larger flakes. Nothnagel 
has reported a jelly-like consistency of a mushy stool in a case of jejunal diar- 
rhea, in which the mucus was not derived from the intestinal wall, but came 
probably from the bile. It is important that one be able to recognize mucus, 
as mistakes have been made in the presence of swollen vegetable tissue, fruit 
pulp, echinococcus membranes, and even of parasites. 

The ordinary form in which mucus appears in the feces is in clumps, 

floccuH, or shreds with irregular margins. These pieces may vary in size from 

those just visible to those several inches in length. In some cases strips, 

tubes, ribbons, or macaroni-like pieces are observed, which are especially 

frequent in enteritis membranacea or mucous colitis. The amount of mucus 

passed may vary from a few flakes to an enormous mass, Bories having seen 

120 grams in one movement. Occasionally one may see, especially in the 

^ Zentralbl. f. Chir., 1914, XLI, 1182; Arch. f. Verdauungskr., 1914, XX, 552. Sec, also, 
Lyle, Curtman and Marshall, Jour. Biol. Chem., 1914, XIX, 445; Zengerle, Med. Klin., 
1914, X, 1795; Indemans, Nederl. Tijdschr. v. Geneesk., 1915, LIX, 2077; Roberts, Jour. 
Am. Med. Assn., 1915, LXV, 244; Couturier, Lyon Med., 1914, XL VI, 313, advocates the 
use of hematin as a reagent for occult blood; Gregersen, Ugesk. f. Laeger, i9i6,LXXVIII, 
1260; Kelly, Jour. Lab. and Clin. Med., 1916, I, 897; Vaughan, Ibid., 1917, 11,437; Wee- 
huizen, Chem Weekblad., 1918, XV, 1521; Graham, Jour. Med. Res., 1918, XXXIX, 15. 



112 DIAGNOSTIC METHODS 

stools of infants, masses resembling cooked sago granules^ or "frog spawn" 
which Kitagawa has identified as mucinous material. 

The consistency of fecal mucus varies from that of a jelly-like mass to one 
having the density of thin leather. The larger the piece the firmer it appears, 
although exceptions do occur. Pure mucus is usually glassy or jelly-like, 
certain inclusions changing its consistency. Cellular inclusions in the mucus 
change it to a paper-like mass, while some specimens appear tenacious due to 
absorption of protein material or to a diminution of its water-content. These 
inclusions, as well as those of fat or bacteria, will diminish the usual transpar- 
ency of the mucus. 

On remaining long in the bowel, mucus may take on the normal brown 
color of the feces due to hydrobilirubin, a dark orange shade from bilirubin, a 
greenish hue from biliverdin, or a red to reddish-brown tinge from blood pig- 
ments. The usual state of the mucus, however, is colorless. 

It may be stated generally that the most of the macroscopically recogniz- 
able mucus of the feces arises from the large intestine. Owing to its easy 
digestibility the mucus from the mouth, stomach, and upper intestines passes 
out only under conditions of great motihty. The secretion of mucus in the 
small bowel is much less than in the large intestine. Of all recognizable forms 
of fecal mucus only the smallest particles arise from the small bowel, and then 
only when they are found in a fluid feces. The higher the secreting point the 
smaller will the particles be, as a rule. These upper intestinal flecks contain 
much detritus of digestion and half-digested cells or free nuclei and crystals 
in cellular form, frequently a few bilirubin granules being observed. 

The mucus from the large intestine, especially from the sigmoid, is large 
in amount, is jelly-like in appearance and has ordinarily no inclusions. In 
cases of mucous colitis the mucus may be passed in the forms of the large 
strips or bowel-casts, appearing as pure transparent material grayish-white or 
bloody in color and having no inclusions. No fecal matter may be present. 
Such a condition is a pure secretion neurosis and becomes a true inflammation 
only when cell-inclusions are observed. It is to be said that mucus, even with 
a small inclusion of pus or blood, does not necessarily point to ulceration. On 
the other hand, one must not judge from the absence of mucus that no catarrh 
exists, as one frequently finds variations in such excretions. 

Microscopic mucus is much rarer than the macroscopic type, although 
both may be present at the same time. Microscopically, mucus appears as a 
structureless mass, characterized by irregular lines running through it and by a 
difiicultly recognizable margin, showing a more or less transparent ground 
substance in which may or may not be found epithelial cells, pus-cells, blood- 
cells, bacteria, protozoa, food remnants, and crystals. If the mucus comes 
from the higher sections of the bowel the food remnants will predominate, 
while in that from the lower bowel the cellular elements are in excess. 

Mucus is stained with difficulty. For a successful stain the reaction must 
be neutral and very little admixture with foreign material is permissible. 
Thionin colors mucin a specific violet, while the other tissue elements are 
stained blue. Methylene blue and methyl violet stain it but slightly, while 

1 Ullmann, Med. Klinik, 1916, XII, 1230. 



THE FECES II3 

other aniline dyes color only the enclosed cells. lodin may give a diffuse 
yellow. 

(7) Pus. 

Macroscopically visible masses of pus of a gray-white color are occasionally 
found in the stools, but these may hardly be distinguished from mucus par- 
ticles without the microscope. If in large amounts, attention should be 
directed to the perforation of an abscess into the bowel. Such pus is more or 
less intimately mixed with the fecal material. The passage of pus from the 
small intestine and even from the cecum is associated with such a marked 
decomposition that it can be recognized only with the greatest difficulty. The 
nuclei may still persist, but these resemble very closely those of the food cells. 

A few isolated leucocytes are usually present in the feces, as a result of 
diapedesis through the mucous membrane. A pure pus excretion is never 
seen in cases of uncomplicated catarrh, but in ulcerative processes of the large 
intestine and in many affections of the small bowel (dysentery, ulcerative 
coHtis, syphiHs, carcinoma, tuberculosis, and typhoid) pus-cells may be found 
in small masses. 

Casein flocculi should be differentiated from pus masses by microscopic 
examination, which will show fat droplets mixed with the albumin. 

(8) Food Remnants. 

The appearance of macroscopic amounts of food remnants in the feces is 
known as lientery and is dependent upon the nature and amount of food, its 
method of preparation, the degree of its mastication, and upon the condition 
of the digestive organs. 

Much less undigested material remains on a meat diet than after a vege- 
table ration. In the former case we may find small bits of bone, cartilage, 
tendon, hair, feathers, skin, fish scales, and connective tissue; while in the latter 
we may observe cellulose-containing cells, such as those of cereal, cotyledonous 
and leguminous vegetables, skins of fruits, nuts, and vegetables, seeds, etc. 

Cooking has a great influence upon the digestibility of any food. Boihng 
seems to be the best method, although much of the nutritive material is taken 
up by the cooking water. The method of roasting is such as to produce a 
more nutritive but a less digestible substance, unless it is carried only to the 
point of slight coagulation of the protein. The outside layers of roasted 
meats seem to be practically indigestible, according to the work of van Ledden 
Hulsebosch. Smoking seems to be the least desirable form of preparing 
meats. Vegetables become more digestible through the cooking process, 
owing to the bursting of the indigestible cellulose membrane. Those vege- 
tables, such as lettuce, cucumbers, onions, turnips, cabbage, and radishes, 
which are eaten raw, appear in the feces absolutely unchanged. 

While great individual differences exist in the power of utilizing digestible 
or undigestible food, yet we must assume that any appreciable residue, espe- 
cially following the Schmidt-Strasburger diet, is pathological. The digestive 
insufficiency may begin in the mouth as a result of too little mastication. The 
importance of thorough mastication has been especially emphasized by 
Fletcher, ' Lack of digestive power of the stomach will have but httle influ- 

^ See, however, Foster and Hawk, Jour. Am. Chem. Soc, 1915, XXXVII, 1347. 



114 DIAGNOSTIC METHODS 

ence on intestinal digestion, providing the pancreatic secretion is sufficiently 
active, with the exception that raw or smoked connective tissue will be 
undigested and will appear in the feces. Lientery is much less frequent in 
motor disturbances of the stomach than in those of the intestines. Perhaps 
the greatest amount of undigested residue is seen in cases of perforation of an 
ulcer or carcinoma of the stomach into the intestine, a direct communication 
being established between the stomach and colon. 

Naturally, in cases of increased intestinal motility, one will find more 
food residue in the feces than under normal conditions. This motor insuf- 
ficiency is especially answerable for the appearance of undigested starch 
remnants, as, even under the most severe conditions, starch digestion is not 
interfered with to any extent in the bowel, providing mastication and prepa- 
ration of food have been sufficient and the succus entericus has not been so 
diminished by catarrhal processes as to permit of fermentation. The direct 
irritation from the hard particles of cellulose may cause an increased peris- 
talsis and, hence, directly lead to lientery. 

Marked disturbance of protein and fat digestion, evidenced by an intense 
lientery, will occur if the pancreatic secretion be insufficient. This is even 
more marked if gastric disturbance coexists. A lack of bile will cause an 
exclusive disturbance of fat digestion, which is seldom manifested by the 
appearance of macroscopic particles of fat, but is characterized by the typical 
clay color of the stool. 

The final factor which influences lientery is the insufficiency of the ab- 
sorptive power of the intestine. This increases the food residue by retarding 
digestion, according to the law that an accumulation of the products of fer- 
ment activity will prevent further action of the ferment, as well as by holding 
within the bowel the products already digested. 

Only in exceptional cases can one judge of the specific factor at the bottom 
of a lientery, a boundary line between normal and abnormal being drawn with 
difficulty. It is, however, important that one be able to recognize food 
remnants both macroscopically and microscopically. The writer recalls a 
case in which the residue of orange and banana pulp was mistaken for a new 
parasite. This recognition of the nature of a fecal residue is especially im- 
portant in the examination of the infantile stool. Normally, the nursing 
child shows no food residue in the feces, but frequently white flecks or clumps 
are observed, which may consist of casein but perhaps more frequently of 
fat or of soaps. The proper recognition of such particles will require both 
chemical and microscopic examination. 

The following discussion of the appearances of the various food residues 
will include both the macroscopical and microscopical examinations, as these 
are inseparable in practical work. 
(^) Protein Residues. 
(a) Muscle Fibers. 

The appearance of muscle fibers in increased amount in the stool is known 

as azotorrhea} The muscle fibers appear as isolated pieces of different size 

1 Also called creatorrhoea. See Wertheimer, Ztschr. f. klin. Med., 191 2, LXXVI, 57; 
Tileston,Arch.Int.Med.,i9i2,IX,S25;also,Fronzig,Ztschr.f.klin.Med.,i9i3,LXXVII,40. 



THE FECES II5 

and shape. The smallest pieces have a circular or oval contour, the medium- 
sized particles are jagged, while the larger masses have parallel sides and 
angular surfaces. These fibers are colored yellow or yellowish-brown by the 
hydrobilirubin, but may be tinged with bilirubin or by foreign pigments. 
The color will depend both upon the amount of pigment and upon the time 
the fibers have lain in the bowel. 

The smaller pieces may be entirely homogeneous, although these as well as 
the larger bits show both transverse and longitudinal striations. If the 
fibers are well digested, the longitudinal striations may be the only ones show- 
ing, and even these may disappear. No nucleus is visible unless the pancre- 
atic secretion is entirely lacking.^ No specific micro-chemical reactions are 
known for these fibers. They may be tested with any of the color reactions 
for protein material. 

Connective tissue and elastic tissue fibers are occasionally associated 
with muscle fiber and may be recognized by their appearance. 

(b) Casein. 

This is found especially in the infant stool and is always pathological. 
These masses, known as curds, are more or less round clumps which vary in 
size from that of a pin head to that of a hazel-nut. They are either pale or 
golden-yellow, the larger masses being a pure white inside. These curds 
always have a distinct yellow tone exteriorly, but the interior is pure white. 
If these particles are pressed between a slide and cover-glass, they spread out 
like white cheese and show absolutely no structure microscopically. These 
masses show the protein reactions. 
Leiner's Test. 

A small amount of fecal matter containing these curds is spread on a slide 
and dried in the air. It is then fixed by heat and stained with a mixture of 
equal parts of a 0.75 per cent, solution of acid fuchsin and methyl greenin5o 
per cent, alcohol (dilute mixture i o times with water) . At the end of 1 5 minutes 
the slides are placed in distilled water and left for one hour. Casein and para- 
casein will take a pale blue or violet color, while similar substances will show 
a greenish tone. 

Microscopically, one may differentiate from these true casein particles 
(curds), certain products which are more or less normal in the stools of the 
child. These masses are smaller than the curds, being not usually over pin- 
head size, are more yellow in color, and appear under the microscope as clumps 
of fatty acid crystals or of fat droplets and bacteria bound together by mucus. ^ 
(B) Fat Residues. 

Fatty substances are present in all stools, if there be any in the diet, either 
in the form of neutral fat, free fatty acids, or soap, especially of calcium and 
magnesium. If present in macroscopically recognizable amounts, the con- 
dition is known as steatorrhea. 

Neutral fat may be present in the form of white colorless clumps of differ- 
ent size and irregular in shape, some being globular while others are distinctly 

^ See Atchley (Arch. Int. Med., 1915, XV, 654), whose experiments tend to disprove the 
value of Schmidt's nuclear test. 

"^ See Courtney, Am. Jour. Dis. Child., 191 2, III, i. 



Il6 DIAGNOSTIC METHODS 

angular. The more usual form is the refractive, opaque, more or less yellow 
globule. Occasionally the fat may appear as a melted oil which hardens 
over the surface of a formed stool or gives the appearance of vaselin to a semi- 
solid stool. Neutral fat may be recognized by its ready solubility in ether 
and its black color on treatment with osmic acid and deep red color when 
acted upon by Sudan III or Scharlach R. 

Fatty acids appear either as scales or as crystals of varying form. The 
scales may not be distinguishable from those of pure fat except by their easy 
solubility in cold alcohol. The crystals are thin, delicate, curved needles, 
which run to a distinct point and are grouped in thick masses. Other types 
of fatty acid needles may occur, such as the small lancet-shaped plate or those 
closely resembling the soap crystal. These crystals are colorless and are 
soluble in cold alcohol, showing no stain with the above reagents for free fat. 
The fatty acid scales are, however, stained by these dyes. 

The soaps likewise appear in the feces as scales or as crystals. The 
scales are less refractive, more firm, usually more angular, and may be color- 
less or yellow-brown from hydrobilirubin or yellow from bilirubin. The 
crystals appear most frequently as uncolored needles, which are shorter, 
plumper, not so pointed as the fatty acid crystals, and are arranged in clusters. 
Schmidt has described a peculiar form of crystal, which he styles the '^ crack- 
nel" form, a round type with a sunken center and a raised border. These 
crystals are insoluble in ether, do not melt on warming, and are uncolored 
with stains. Treated with acids they form fatty acid crystals. 

Any condition which interferes with the proper absorption of fat by the 
mucosa or lymphatics will lead to steatorrhea. This condition may be ob- 
served, physiologically, after the ingestion of large amounts of fat, as in the 
oil treatment of gall-stones, but we are little concerned with such findings. It 
is seen, pathologically, in cases of atrophy of the mucosa, in amyloid disease 
of the intestines, tuberculous ulceration, tubercular peritonitis, tabes mesen- 
terica, and even in catarrhal enteritis. 

The peculiar, glistening, gray-white, pasty, acholic stool of steatorrhea is 
seen more frequently, however, in cases of biliary obstruction and of pancre- 
atic disease. The fat in these cases of biliary stasis is in the usual form, that is 
three-fourths of the ether extract of the bile-free feces is present as fatty acids 
and soaps and one-fourth as neutral fat. In cases of acholia 50 to 80 per 
cent, of the fat will be unabsorbed instead of the normal 5 to 10 per cent.^ 

In the fatty stools of pancreatic disease, the fat is largely present as fatty 
acid, no soap being found. The association of steatorrhea with the absence 
of decomposition products, few bacteria, and the presence of maltose in the 
feces is much more indicative of pancreatic disease than is steatorrhea alone 
(Le Nobel). 

(C) Carbohydrate Residues. 

Starch may be present, in the normal feces, enclosed within plant cells 
which have resisted digestion, but well-preserved starch granules are never 

^ See von Hoesslin and Kashiwado, Deutsch. Arch. f. klin. Med., 191 2, CV, 576; Avir- 
agnet and Dorlencourt, La Nourrison, 1919, VII, 283. 



THE FECES II7 

found normally. A few partially digested or complete colorless granules may 
indicate no abnormality, but many will point to a hyperacidity or to a disturb- 
ance in the small intestine, especially to an insufficiency of the succus en- 
tericus leading to the so-called "fermentative dyspepsia" (Schmidt). 

Starch may be detected by the addition of Lugol's iodin solution to a 
small portion of the feces spread upon a slide. A blue coloration will in- 
dicate starch, while a red tone will show the presence of erythrodextrin. 

The varieties of cellulose- containing substances found in the feces are as 
numerous as are the types of such food material. These have been discussed 
previously and cannot be here elaborated. Certain microchemical tests may 
prove valuable in the identification of such cellulose material. 

Cellulose treated with sulphuric acid and then with iodin solution gives 
a blue color due to the conversion of cellulose into amyloid. 

If a solution of zinc chlorid be allowed to act upon a suspected mass of 
cells, which have been previously treated with Gram's iodin solution, a purple 
coloration will be produced. If a neutral or weakly alkaline solution of Congo- 
red be added to cellulose, it will be stained a distinct red. Cellulose is soluble 
only in an ammoniacal solution of cupric oxid, known as Schweitzer's reagent. 
(9) Biliary Constituents. 

As a rule, the unchanged biliary acids, glycocholic, taurocholic, and 
cholalic acids, are absorbed from the bowel so that they do not appear in 
appreciable amounts in the feces. Schmidt believes that cholalic acid is 
present in slight amounts in all feces and that all the bile acids may be in- 
creased in pathologic conditions. 

Bile pigments, on the other hand, are always present, chiefly in the form 
of hydrobilirubin, although bilirubin may occur. In pathologic conditions 
we may find, beside these two pigments, biliverdin, bilifuscin, bilicyanin, and 
bilihumin. Stercobilin (urobilin) in the feces is usually an index of blood dis- 
integration.^ The tests for these pigments as well as for the free bile acids 
and their salts will be considered in detail in the section on Urine. ^ 

In case of cholelithiasis gall-stones of varying size may be found in the 
feces, although not in every case. It is, therefore, of the greatest importance 
, that the feces be carefully examinedTin suspected cases for the presence of these 
concretions. This is especially the case whenever a severe, cohcky, abdomi- 
nal pain of doubtful origin exists. The feces should be well mixed with 
rater and passed through a fine sieve which will retain any of the suspected 
particles. A single examination of the feces is not sufficient in such cases 
.unless the stones be found. In cases of negative findings, the stool should be 
searched for at least two weeks following a suspected attack. According to 
'Naunyn, only the very firm stones will leave the bowel, the softer ones break- 
ing up into small bits in the bowel. 

These gall-stones vary from the size of a pin-head to that of a pigeon's egg 
and are found as small crumbling masses or as hard stones which either have a 

^ See Robertson, Arch. Int. Med., 1915, XV, 1072; Ibid., 1915, XVI, 429. 

2 See, however, Jovine, Gazz. d. osp., 1913, XXXIV, 1071. For the chemical composi- 
tion of human bile consult Czyhlarz, Fuchs and von Fiirth, Biochem. Ztschr., 1913, XLIX, 
120; also, Rosenbloom, Jour. Biol. Chem., 1913 XIV, 241. 



Il8 DIAGNOSTIC METHODS 

jagged surface or a smooth surface with characteristic facets, indicating the 
presence of many such stones. These stones consist for the most part of 
cholesterin mixed with the biHary pigment, or of compounds of calcium with 
the biliary pigments. These calcareous stones may be combinations of the 
various bile pigments and are always the hard facetted type, while the choles- 
terin stones are softer and may be colorless or tinged with bile pigment. The 
nucleus of these stones is usually a mass of organic detritus, in some cases 
being made up of clumps of bacteria, such as the typhoid bacillus and bacillus 
coli communis. A gall-stone may be usually recognized from its fractured 
surface, but frequently it becomes necessary to submit it to chemical examina- 
tion for purposes of identification. 

In this examination for gall-stones the worker must not be deceived by 
the presence of extraneous substances such as seeds, cherry-stones, fats and 
soaps of high melting point and of masses of impacted feces. In the treat- 
ment of gall-stones by the use of olive oil many of these soft fatty translucent 
masses appear in the feces which are in no way associated with gall-stones. 

(lo) Intestinal Sand and Concretions. 

By intestinal sand we have reference to the small granules or masses of 
inorganic salts which appear in the feces. These masses are very small, may 
be spherical or irregular, are usually hard, usually have a reddish-brown or 
green color and consist of inorganic compounds mixed with organic detritus, 
especially with fat and bacteria. While most of this material is made up of 
ingested substances, we may have in cases of neurasthenia and especially in 
those associated with mucous colitis the excretion of large numbers of these 
particles as the result of a true secretory neurosis. Myer and Cook^ have 
recently shown that the banana may be a source of this intestinal sand.^ 

The massing together of this intestinal sand may form intestinal concre- 
tions or enteroliths. As a rule, however, these intestinal concretions have as a 
nucleus some foreign body around which calcium and magnesium salts of 
phosphoric and carbonic acid or ammonium magnesium phosphate have been 
deposited. These enteroliths are usually hard, heavy, and round, being 
colored, as a rule, brownish. ^ A second form of intestinal calculus, known as 
coproliths, are irregular in shape, usually softer than the enterohths, and consist 
of inorganic material, mixed with inspissated feces. Neither of these types of 
intestinal concretion have any great pathologic significance, although the 
possibility must be granted that some of them might either form or lodge 
in the appendix and thus be accountable for an acute appendicitis. 

(ii) Tissue Fragments. 

The examination of feces for tissue fragments is more or less unsatisfactory, 
owing to the fact that thdse fragments are difficultly recognizable after being 
partially digested by the juices of the intestinal canal. It not infrequently 
happens, however, especially in carcinoma of the lower bowel, that such f rag- 

1 Am. Jour. Med. Sc, 1909, CXXXVII, 383. 

^Talbot (Jour. Am. Med. Assn., 1913, LXI, 238) shows that the banana is not always 
the source. 

3 See Coerr, Jour. Am. Med. Assn., 1913, LXI, 2238. 



I 



THE FECES II9 

ments may be obtained and a diagnosis made possible by microscopic exami- 
nation. A diagnosis of malignancy would better not be made unless these 
tissue fragments show typical cellular arrangement, or at least, typical ar- 
rangement of the nuclei. The writer has seen a diagnosis of carcinoma made 
on the basis of a strip of mucus with cell enclosures and would, therefore, warn 
the worker to be on his guard against the possibility of such an absurd mistake. 

III. Microscopic Examination 

This examination includes the search for many substances which have been 
described under the macroscopic examination, so that these need not be here 
considered. There are, however, some elements, both morphological and 
crystalline, which have not been treated. 

For any success whatever in the microscopic examination of the feces, 
great care must be taken in the selection of ihe material for examination. 
This is especially true when searching for parasites or their ova, as well as for 
the differentiation between various food particles and secretions from the 
. intestinal wall. If the stool be soft and mushy it should be thoroughly mixed 
by stirring and suspicious particles looked for macroscopically. Fresh speci- 
mens as well as stained preparations are then made and examined both with 
the low and high power. In examining feces for bacteria it is always well to 
mix the feces with water and centrifuge for a few minutes. The bacteria will 
remain suspended in this process and the coarser fecal material will be depos- 
ited. The fluid is then poured off and mixed with equal parts of alcohol, 
the mixture being again centrifuged when the organisms will be found in the 
sediment. This procedure is the one adopted by Strasburger in estimating 
the proportion of bacteria in the dry stool. 

Various microchemical reactions are carried out with isolated fecal 
material and frequently are of great importance. In this procedure a few 
drops of reagent are allowed to flow under a cover-glass which covers the 
specimen. The staining material may be drawn under the cover-glass by 
placing a piece of ordinary filter-paper on the opposite side of the cover-slip. 

If the examination for amebae is to be made it is essential that the stool 
be kept warm, as the characteristic differentiating point of an ameba is its 
active motihty. This can be done by placing the feces in a bottle which is 
surrounded by warm water until the examination is made. It is wise, also, 
to make the examination upon a warm stage, although this is not absolutely 
essential unless extended search has to be made for the parasite. There are 
no specific stains for the feces, the ordinary Lofifler's methylene blue serving 
very well for a general staining of bacteria, while characteristic stains must be 
used when searching for special organisms. 

Morphological Elements. 

Epithehal cells are present in every specimen of feces. These may be 
squamous in form and are usually found in the mucoid particles of the stool. 
They are especially present in rectal carcinoma and in ulcerative conditions 
of the lower bowel. The cylindrical type of epithelium is much the commonest 
form and is found, also, in association with the mucus. Many well preserved 



I20 DIAGNOSTIC METHODS 

cells may be present if their source is in the lower bowel, but, as a rule, these 
cells show all types of degeneration. They occur in catarrhal inflammations 
of the intestinal mucosa and are rarely associated with pus-cells unless ulcer- 
ation has taken place. If the irritation is in the small bowel these cells are 
always more or less digested and contain bilirubin particles in cellular arrange- 
ment, while if from the lower bowel the cell is usually intact. 

The presence of red blood-cells is determined more by the chemical ex- 
amination than by the microscopical. It may occasionally happen that these 
cells may be seen, but a good result will depend upon a fortunate selection of 
the material for examination. A few scattered leucocytes are practically always 
found in the feces, ^ and assume pathologic importance only when present 
in large amounts as pus-cells, whose occurrence has been previously discussed. 
Crystals. 

Besides the crystals of free fatty acids and soaps previously mentioned 
we find large numbers of other crystals in the feces. Among these we observe 
neutral phosphates of calcium and of magnesium which appear as wedge- 
shaped crystals occasionally forming rosettes in the former case while in the 
latter the crystals are more in the form of rhombic plates. The ammonium 
magnesium phosphate crystals are practically always present in the feces, 
appearing either as the typical coffin-lid crystal or as irregular fern-like masses. 
These triple phosphate crystals were at one time supposed to be characteristic 
of typhoid stools, but like so many other things their importance has been 
exaggerated. Various crystals of calcium compounds are observed, such 
as calcium carbonate, calcium sulphate, and calcium oxalate, along with 
calcium salts of unknown fatty acids, which have been described as irregular, 
oval, or circular masses, either fissured or showing concentric striations, and 
being always bile-stained. The lactate of calcium is seen in the form of 
radiating needles arranged in sheet-like masses in the stools of children on a 
milk diet. 

Cholesterin crystals are found as thin, transparent, rhombic plates with 
notched corners. These crystals do not always appear in the typical shape, 
so that they should be tested by the addition of concentrated sulphuric acid 
when the cholesterin crystals will change from a yellow to a blood-red, violet, 
green, and finally blue color. The Charcot-Leyden crystal appears in typical 
form in the feces as a colorless, diamond, double pyramid-shaped crystal. The 
presence of these crystals is practically characteristic of helminthiasis, al- 
though they may indicate the presence of any parasite from the least harmful 
to the most pernicious. Hematoidin crystals occur as reddish-yellow rhombic 
plates or as groups of needles or amorphous masses in stools showing the 
presence of blood chemically. These have no special significance and are not 
found as frequently as are the other blood pigments. 

After the use of bismuth preparations we find the oxid of bismuth appear- 
ing in the form of black irregular rhombic crystals with notched edges. Char- 

1 Albu and Werzberg (Ztschr. f. klin. Med., 1912, LXXIV, 394) call attention tr 
the frequent presence, in the feces of amebic dysentery, of eosinophile leucocytes and 
myelocytes. See, also, Barnett, Arch. Int. Med., 191 7, XIX, 695; Lynch, Jour. Lab. 
and Clin. Med., 1917, II, 251. 



» 



THE FECES 121 

coal appears in the form of irregular black masses which are larger and not so 
rhombic as are the bismuth crystals. 

The examination of the feces for the various bacteria as well as the para- 
sites and parasitic ova will be discussed in a special section. Various sub- 
stances appearing in the feces have been mistaken for parasites or their ova. 

IV. Chemical Examination 

The chemical examination of the feces would naturally embrace both the 
qualitative and quantitative estimation of the products of digestion and de- 
composition as well as the estimation of the undigested portion of the food 
along with the products derived from the gastrointestinal canal itself. Clin- 
ically, such work is rarely carried out and has questionable value from a 
diagnostic standpoint. However, in following the metabolism in any special 
case it is essential that the absorbed and unabsorbed portion of the food be 
known. This applies more particularly to the proximate food principles, 
protein, fat, and carbohydrate, although in some cases the estimation of the 
inorganic intake and excretion is of first importance.^ The writer cannot 
attempt in the scope of this work to go into great details regarding the chem- 
ical examination of the feces, but must limit himself to a few selected topics. 
Reaction. 

The normal reaction of the feces does not vary much from a neutral one, 
although it has a tendency to be slightly alkaline, owing to the presence of the 
alkaline secretions of the intestinal tract. ^ The alkalinity of these secretions 
is diminished both by the combination of the alkali salt with the digestion 
products and by the absorption of some of this alkaline material into the 
blood. This reaction may at times be acid owing to the formation of lactic 
and butyric acids in the fermentative processes, while an increased alkalinity 
may be observed in cases of markedly increased ammoniacal decomposition. 
A pure meat diet gives, as a rule, an alkaline feces, while a pure carbohydrate 
or fat diet will have a tendency to form an acid feces. 

The reaction of the feces under pathologic conditions is of little impor- 
tance, although in typhoid fever the reaction is somewhat more strongly alka- 
line than in almost any other condition. So much depends upon the diet as 
well as upon the condition of the digestive organs that no conclusions at 
present may be drawn from the reaction of the feces. 
Total Solids. 

The normal amount of feces passed in 24 hours ranges from 100 to 250 
grams, of which about 75 per cent, is water and 25 total solids. Although the 
determination of the total solids of the feces has little of importance in itself, 
it is essential that one should know the dry weight of the feces in order that he 
may properly calculate the amount of the various substances in the dry feces. 

In determining the total solids in the feces, the specimen, preferably the 

24-hour specimen or each movement separately, is placed in an evaporating 

dish, covered with a small amount of alcohol, and heated over a water-bath 

^ See Holt, Courtney and Fales, Am. Jour. Dis. Child., 1915, IX, 213; von der Heide, 
Biochem. Ztschr., 1914, LXV, 363. 

2 See Bruce, Jour. Lab. and Clin. Med., 1919, V, 61. 



122 DIAGNOSTIC METHODS 

with frequent stirring. Small amounts of alcohol should be added as 
evaporation proceeds in order to hasten the drying process. It is, moreover, 
necessary to add a small amount (lo to 15 c.c.) of dilute sulphuric acid and to 
mix it thoroughly with the feces. This combines with the free ammonia, 
forming a non-volatile salt, and thus prevents loss of nitrogen. If the stool 
be rich in fats, it is wise to add a weighed amount of dry washed sand to make 
the mass more porous and thus permit of quicker drying. When the water 
has been driven off on the water-bath, the specimen may be placed in the dry- 
ing oven at 105° and left for several hours, after which it is placed in the desic- 
cator and dried to constant weight. Knowing the weight of original substance 
taken and the weight of the dry substance, it is very easy to determine the 
percentage of total solids. 

An increased total soUds of the feces is observed in most cases of constipa- 
tion, while in diarrhea the solid residue is much diminished. Increased sepa- 
ration of fluid into the bowel and diminished absorption from the bowel are 
the factors regulating the fluid content. Nothing can be determined, how- 
ever, by the examination of the amount of total solids, as regards the patho- 
logic conditions accountable for an increase or a decrease. 

Total Nitrogen. 

The determination of the total nitrogen must always be made in estimating 
the metabolism in any given case. In so doing it is likewise essential that one 
should know the absolute nitrogen intake, as without such a factor nothing can 
be learned from the excretion either in the urine or in the feces. The daily 
excretion of nitrogen in a fasting condition varies from i to 4 grams, on a 
mixed diet this may run from 3 to 7 grams, while on a vegetable diet the nitro- 
gen of the feces may be as high as 10 grams, a pure meat diet yielding between 
2 and 6 grams. In pathologic conditions this amount is practically always 
increased, due both to lack of digestive power and to decrease in the absorp- 
tive function of the intestines. Outside of metabolic experiments the absolute 
amount of nitrogen of the feces has no great diagnostic importance beyond 
showing some perversion of intestinal activity. ^ In insufficiency of the pancre- 
atic secretion, we are apt to find the greatest loss of nitrogen by way of the feces. 

The constituents which go to make up this total nitrogen are the various 
undigested protein bodies, such as albumin, globulin, nucleoprotein, nucleo- 
albumin, and gelatin, along with partially or completely digested products of 
protein origin, bacteria, and secretions from the intestines. These latter 
bodies embrace albumoses, peptone, the various amino-acids, and the hexone 
bases along with a certain amount of ammonium salts. The chemical prop- 
erties of these substances, as well as the methods of their chemical detection 
and estimation, must be looked for in works on physiological chemistry. The 
bacteria form a large percentage of the total nitrogen. ^ It is rare, even in 
metabolic work, that the nitrogen partition of the feces is determinefd. 

1 See McCrudden and Fales, Jour. Exper. Med., 191 2, XVII, 20 and 24; also Koplik and 
Crohn, Am. Jour. Dis. Children, 1913, V, 36; Gamble, Ibid., 1915, IX, 519; Van Slyke, 
Courtney and Fales, Ibid., 533. 

2 Mattill and Hawk (Jour. Exper, Med., 191 1, XIV, 433) shows that the bacterial nitro- 
gen constitutes 53.9 per cent, of the total fecal nitrogen. 



THE FECES I 23 

Much more importance attaches, with our present knowledge, to such division 
of the nitrogenous material of the urine. Likewise the products of abnormal 
decomposition of protein material, taking place in the bowel with the forma- 
tion of such products as in.dol, skatol, and phenol, are rarely searched for in the 
feces. ^ Our clinical knowledge of the importance of these substances is con- 
fined largely to their detection and estimation in the urine. In the study of 
the metabolism of various cases of cystinuria, which is associated with abnor- 
mal protein disintegration, certain diamines, such as cadaverin and putrescin, 
have been isolated from the feces by Udransky and Baumann.^ 
Fat. 

The chemical estimation of the amount of fat is of importance only in 
metabolic work, as in the more direct clinical examinations this substance is 
detected by macroscopic and microscopic methods. It is, however, essential 
that the loss of fat by way of. the feces should be known before a proper meta- 
bolic balance can be struck. 

The dried feces is treated with a small amount of i per cent, acid-alcohol 
and evaporated to dryness in order to convert any soaps which may be present 
into the fatty acids. The dry residue thus treated is then placed in a Soxhlet 
apparatus and extracted with ether for at least 72 hours. The ether, which 
has taken up the free fat and the fatty acids, is then evaporated and the resi- 
due of fats weighed. From this weight the percentage of fat may be deter- 
mined, and, knowing the original dry weight of the total 24-hour feces, the 
total fat lost by way of the feces may be readily calculated.^ 

The amount of fat in the feces will depend much upon the amount and 
quahty of the diet. In fasting conditions the amount is usually about i gram. 
On diets poor in fat the amount in the feces may exceed that of the diet, thus 
indicating a loss of fat from the body. It seems to be a general rule that the 
higher the melting point of the fat of the diet the greater will be the loss in 
the feces. Thus on a diet containing only butter as a fat about 4 per cent, of 
the intake will be lost, while with pork fat the loss may be as high as 13 per cent. 
It is impossible to say, a priori, just what the fat content of^a normal feces 
would be. With the ordinary diet of our country it would range from 2 to 7 
per cent, of the intake, which should be somewhere about 100 grams of fat per 
day, representing a loss of from 2 to 7 grams daily. 
Carbohydrates. 

As previously stated, carbohydrates may appear in the feces either in the 

form of starch or of cellulose. Only in exceptional conditions do we find any 

of the monosaccharids present. Disaccharids, such as lactose and maltose, 

may be occasionally found, but only when there is a combination of insuffici- 

^ See Moewes, Ztschr. f. exp. Path, und Ther., 191 2, XI, 555. See, also, Rodella, Arch. f. 
Verdauungskr,, 1914, XX, 657; Whipple (Jour. Am. Med. Assn., 1915, LXV, 476) believes 
that the intoxication of intestinal obstruction is due to the absorption of a primary proteose. 

2 See Hopkins (Arch. Int. Med., 1913, XI, 300) for a discussion of the presence of hemo- 
lysins in fecal extracts. 

3 See Cowie and Hubbard, Am. Jour. Dis. Child., 1913, VI, 192; also, Bloor, Jour. Biol. 
Chem., 1913, XV, 105; Ibid., 1913, XVI, 517; Saxon, Ibid., 1914, XVII, 99; Gephart and 
Csonka, Ibid., 1914, XIX, 521; Smith, Miller and Hawk, Jour. Biol. Chem., 1915, XXI, 
395; Laws and Bloor, Am. Jour. Dis. Child., 1916, XI, 229; Dyer, Jour. Biol. Chem., 1917, 
XXVIII, 445; Holt, Courtney & Falls, Am. Jour. Dis. Child., 1919, XVII, 38. 



124 



DIAGNOSTIC METHODS 



ency, both of a salivary and pancreatic ferment. The presence of any ap- 
preciable amount of starch granules must be considered pathological, while 
the amount of cellulose will depend upon the amount in the food as well as 
upon the preparation of the food and its mastication. 

Nothing is to be learned, from the clinical standpoint, by the determina- 
tion of the absolute amount of carbohydrate in the feces. In the study of the 
utilization of food substances by the system and in general metabolic work 
it is, however, necessary to know just how much of the carbohydrate intake is 
absorbed. This may be determined indirectly by subtracting from the 
weight of the dry feces the sum of the protein, fat, and ash. This result will 
represent a much higher figure for carbohydrates than the one obtained by 
direct determination. In the direct determination much difference exists 
between the soluble forms, and the insoluble cellulose. In the latter case we 
have to do with a useless form of carbohydrate and should, therefore, direct 
our attention rather toward the estimation of the amount of undigested or 
partially digested starch. We should determine the amount of cellulose and 
subtract this factor from the total carbohydrate obtained in the previous 
subtraction. This corrected figure will represent more nearly 
the true amount of carbohydrate than will the former, al- 
though for scientific purposes a direct determination is 
essential. 

The starch may be estimated by taking a weighed 
amount of the dry feces and treating with 50 c.c. of 10 per 
cent. HCl. This is then boiled for one-half hour in order to 
convert the starch and digested portions of starch into the 
monosaccharids. This acid solution is then filtered and 
washed with sufhcient water to make the total approximately 
the same as that of the original solution. The filtrate is 
then neutralized with sodium hydrate and made up to exactly 
100 c.c. in a volumetric flask. The sugar in this solution 
is then determined by the methods outlined under Urine. 
This method, although not absolutely accurate, will yield 
results sufficiently correct for ordinary purposes. More 
accurate results will be given by the Volhhard-Pfiuger method 
to be discussed later. The amount of glucose, as deter- 
mined by either of these methods, will give, if multiplied by 
0.94, the amount of starch in the feces taken . The percent- 
age may then be readily calculated and the total quantity 
of the 24-hour excretion of starch determined. 

The fermentation method of Schmidt was advanced to 

permit of rough estimation of the presence of pathological 

amounts of starch, which, under ordinary circumstances, 

should have been digested. The principle of the method is 

the estimation of the amount of gas produced by the action of the intestinal 

bacteria upon the sugar which is formed from the starch by the intestinal 

amylolytic ferments. Prior to this determination the patient is placed upon 



Fig 21. 
Schmidt's fermen- 
tation apparatus. 



THE FECES I 25 

the test diet outlined on page 100. The test is made as follows: Approxi- 
mately 5 grams of the moist stool are placed in the vessel a which is then 
filled with water and the contents thoroughly mixed (see cut) . The stopper is 
then placed in the fermentation flask in such a way that no air-bubbles are left. 
The tube h is filled with tap-water and closed with a small stopper without the 
inclusion of any air-bubbles. The tube c is now placed in position as show^n 
in cut and the whole apparatus put in an incubator for 24 hours. The tube c 
has a small opening in the top so that water may be readily forced from the 
tube h into c by pressure of the gas produced. According to Schmidt o.i 
gram of starch will cause the tube h to show about one-half its volume of gas. 
Normally, a positive result is said to occur when the tube is one-fourth to one- 
third filled with gas. The material in the fermentation flask a should be 
tested with litmus-paper after the test is complete to obtain the reaction of the 
mixture. If the gas formation is, as it should be, due to carbohydrate fermen- 
tation there will be a slight increase in the acidity of the mixture, while if it be 
due to protein putrefaction the reaction will show a slightly increased 
alkalinity. 

The various decomposition products of carbohydrate digestion and fer- 
mentation are rarely of importance in fecal examinations. These products 
consist of volatile fatty acids, lactic acid, saccharic acid, alcohol and aldehyde, 
and the conjugated glycuronic acids. Tests for these various substances will 
be found in various parts of this work or in works on physiological chemistry 
to which the reader is referred.^ 

From the clinical standpoint the gases produced by the processes of fer- 
mentation and putrefaction in the bowel have little value. The same is to be 
said of the quantitative estimation of the inorganic constituents of the feces. 
These latter may be determined by methods outlined in quantitative analysis. 

The various ferments found in the intestinal canal may be detected in the 
feces, but such examinations are at present of little clinical value. . Methods 
of isolating these ferments and of determining their activity belong more to 
physiological chemistry than to clinical diagnosis and will, therefore, be 
neglected. 2 

Phenoltetrachlorphthalein Test. 

There is no question but that the liver has a more varied and manifold 
functional activity than any ather organ of the body. Slight disturbances in 
hepatic activity are a matter of relatively frequent occurrence, but these are 
not attended with any marked perversion of function as the larger portion of 
the parenchyma of the liver may not be involved. In disease, however, of the 
liver the character of the functional changes will vary with the nature of the 
underlying pathological condition. There is not so much difficulty in deter- 
mining, from clinical and laboratory data, that hepatic disease exists as there 
is in estimating the intensity of the disease and the degree of disturbance of 
the functional activity. For this reason various attempts have been made to 

^See Fischer, Ztschr. f. exper. Path. u. Therap., 1913, XIV, 179. 

2 See Crohn, Am. Jour. Med. Sci., 1913, CXLV, 393; also, Chace and Myers, Arch. Int. 
Med., 1913, XII, 628; Brown, Bull. Johns Hopkins Hosp., 1914, XXV, 200; Einhorn, Am. 
Jour. Med. Sc, 1914, CXLVIII, 490; Crohn, Ibid., 839; Arch. Int. Med., 1915, XV, 581. 



126 DIAGNOSTIC METHODS 

devise tests which would indicate such functional variations sharply and per^ 
mit of a judgment concerning their intensity. While many of these tests have 
been proven of questionable value, some of them, such as the levulose and 
galactose tolerance test as well as the urobilinogen estimation, are of some 
importance in diagnosis and prognosis and will be discussed in the section on 
Urine. These tests have, for the most part, lacked a quantitative element, 
indicating more the presence than the extent of the disease. 

Rowntree, Hurwitz and Bloomfield have introduced a test for the functional 
activity of the liver, which is probably the best at our disposal. They take 
advantage of the fact, discovered by Abel and Rowntree, that phenolte- 
trachlorphthalein, a drug first prepared by Orndoff and Black, escapes from 
the body only in the bile and may be recovered from the feces to a consider- 
able extent. Under normal conditions the drug is not excreted to any ap- 
preciable extent in the urine. In other words, the liver exerts a specific action 
in excreting this dye, just as does the kidney in eliminating phenolsulphone- 
phthalein. With this fact as a basis, these workers were able to show that a 
decreased capacity for its excretion was a resultant of lowered functional 
activity due to disease, and that minimal amounts of the dye escaped with the 
urine in pathological conditions of the liver. By direct estimation of the 
amount of this dye excreted by the bile and carried into the intestines one 
may readily estimate the presence and, to some extent, the degree of the 
functional disturbance of the liver. 

The solution used for the injection is an aqueous solution of the disodium 
salt of phenoltetrachlorphthalein. This is prepared as follows: 2.5 grams of 
the phenoltetrachlorphthalein are placed in a 200 c.c. flask with 5 c.c. of an 8 
per cent. (2N) sodium hydrate solution and 45 c.c. of freshly distilled water. 
Boil for 20 minutes under a reflux condenser. Filter the solution into a 100 
c.c. flask, when it is ready for use. It is an intense purplish-red solution, 
which does not keep for many days owing to the precipitation of the dye by 
the CO2 of the air. Hence relatively fresh solutions must be used. 
Method of Administration. 

Eight c.c of the above solution, representing 400 mg. of phenoltetrachlor- 
phthalein, are administered intravenously to the patient, using all the anti- 
septic and aseptic precautions which this method involves. The funnel and 
tubes are filled with sterile physiologic salt solution and, after the flow is es- 
tablished, the solution of the dye is added. Fifty to 100 c.c. of salt solution 
are used, the dye being washed in with the salt solution until the fluid enter- 
ing the vein is colorless. 
Collection of Material. 

Before beginning the administration of the dye, active purgation of the 
patient should be induced and should be continued throughout the time of 
observation. If little feces can be obtained, enemata should be employed. 
Collect all the fecal material which passes for 48 hours and the urine for 24 
hours. 

Place the total 48-hour specimen of feces in a large bottle and dilute it with 
I to 1.5 liters of water, depending on the amount of feces. Place this mixture 



THE FECES I 27 

in a shaking machine for 5 to 20 minutes. Without allowing time for sedi- 
mentation, one- tenth of the total mixture is placed in a liter flask and to this is 
added 5 c.c. of 40 per cent, sodium hydrate, which causes the mixture to take 
on a dirty red color. Now dilute the mixture to i liter with water, stopper the 
flask, and shake thoroughly. One hundred c.c. of this preparation are placed 
in a 200 c.c. flask and 5 c.c. of a saturated solution of basic lead acetate are 
added. Now add 5 c.c. of 40 per cent, sodium hydrate solution to elicit the 
maximum intensity of red color. If the amount of NaOH added does not 
seem sufficient, add a little more, being careful not to redissolve the precipi- 
tate present.^ Make up the contents of the flask to 200 c.c, shake and filter a 
small portion for comparison with a standard solution of phenoltetrachlor- 
phthalein. This comparison is made in a colorimeter, either of the Duboscq 
type or of the type of Rowntree and Geraghty's modification of the colori- 
meter of Autenrieth-Konigsberger. For comparison one may use 0.4 c.c. of 
the original solution, making this up to a liter and adding enough NaOH to 
insure the maximum color. 

There are certain elements connected with this test that are not very de- 
sirable. It involves intravenous injection, which is followed in many cases 
by thrombosis at the site of injection. This is not sufficient to occasion 
serious discomfort, but the fact should be remembered and its possibilities 
taken into consideration. Further, it necessitates a careful collection of 
feces for 48 hours. The quality of the red color obtained in the fecal extract 
in some cases is such that accurate quantitative determinations are difficult. 

The results with this test show that a phenol tetrachlorphthalein output in 
the feces below 30 per cent, or its appearance in the urine is quite infrequent in 
health. The output of a less amount is relatively frequent in hepatic disease, 
although a normal output of 30 per cent, does not exclude liver lesions. The 
output of the dye in the feces is independent of the quantity of bile excreted, 
although it is evident that none of the dye will reach the intestines in cases of 
obstruction of the duct. Functional changes, as evidenced by a lessened out- 
put of the dye in the feces, have been most marked in cirrhosis, carcinoma and 
in cachexias associated with severe anemias. Chronic passive congestion 
does not give rise to a much changed excretion, although marked hepatic con- 
gestion associated with insufficiency of the myocardium is usually accompanied 
by a lowered output. Although this test is not to be regarded as diagnostic 
in all cases of perversion of hepatic activity, yet it offers much evidence which 
can be obtained by very few other tests so far advanced. 2 

1 If difficulty is experienced on account of the quality of the color at this point, add to the 
100 c.c. of mixture 5 to 10 c.c. or more of a calcium chlorid mixture (CaCU, 90 grams; cone. 
NH4OH, 10 c.c; water, 50 c.c.) until the best color appears. Dilute to 200 c.c, allow the 
mixture to stand from i to 24 hours and decant the supernatant fluid for comparison. 

2 Orndoff and Black, Jour. Am. Chem. Soc, 1901; XLI, 349; Abel and Rowntree, Jour. 
Pharmacol, and Exper. Therap., 1909, I, 233; Whipple, Mason and Peightal, Bull. Johns 
Hopkins Hosp., 1913, XXIV, 207; Rowntree, Hurwitz and Bloomfield, Ibid., 327; Whipple, 
Peightal and Clark, Ibid., 343; Whipple and Christman, Jour. Exper. Med., 1914, XX, 297; 
Chesney, Marshall and Rowntree, Jour. Am. Med. Assn., 1914, LXIII, 1533; Sisson, Arch. 
Int. Med., 1914, XIV, 804; Whipple and Speed, Jour. Exper. Med., 1915, XXI, 203; 
McLester and Frazier, Jour. Am. Med. Assn., 1915, LXV, 383; Kahn and Johnston, New 
York Med. Jour., 1915, CII, 848.; McNeil, Jour. Lab. and Clin. Med., 1916, 1, 822; Foster 
and Kahn, Ibid., II, 25. 



128 DIAGNOSTIC METHODS 



V. Bacteriology of the Feces 

The bacteria of the intestinal canal are of many types, many of which are 
purely saprophytic, while others may or may not be pathogenic.^ The 
number of these bacteria is usually enormous. One may determine the 
amount of these bacteria by the method of Strasburger, who uses the following 
technic: Two c.c. of feces are rubbed up in a porcelain mortar with 30 c.c. 
of }/2 per cent, hydrochloric acid. This mixture is then placed in centrifugal 
tubes and whirled for one minute. The bacteria will remain in suspension in 
the liquid which is poured off from the sediment. The sediment is again 
rubbed up with a little hydrochloric acid and again centrifuged, the liquid 
being added to the first portion. This procedure may be repeated until the 
fluid no longer becomes turbid on centrifuging. This acid solution, holding 
in suspension the bacteria, is then mixed with an equal portion of ordinary 
alcohol and placed in a beaker which is allowed to remain on a constant 
water-bath at 40° for 24 hours. At the end of this period a portion of the 
fluid may be evaporated and more alcohol added. This mixture is then 
placed in the centrifuge tubes and whirled for several minutes. The bacteria 
are now deposited and the supernatant fluid is poured off and mixed so that it 
may be again centrifuged. The bacterial sediment is washed with alcohol 
and ether in the centrifuge tube and is then placed in a weighed dish. This is 
dried at 100° and again weighed. In order to determine the amount of 
bacteria as compared with the total feces, the dry weight of 2 grams of the 
fresh feces is determined as previously outHned. Knowing these factors, 
the percentage of bacteria in the dry feces may be easily calculated. Stras- 
burger found that this was approximately one-third the weight of the dry 
stool and represented about 8 grams per day.^ As this dry feces is used for 
the determination of total nitrogen, we must bear in mind that the bacteria 
of the feces will represent about one-half of the total nitrogen of the feces. 

It is to be said that the bacterial flora of the intestine is so varied that very 
careful and long-continued work is necessary to isolate a special organism. 
From the standpoint of preventive medicine it should always be remembered 
that the feces contain large numbers of typhoid bacilli and cholera spirilla, so 
that measures should betaken to treat properly suchejecta as soon as voided. 
While the majority of the bacteria of the intestines are saprophytic, being in- 
troduced with the food or drinking-water, yet we find the Bacillus coli 
communis as a normal habitant of the intestine. This organsism is usually 
harmless, but may, under certain conditions, become distinctly pathogenic, 
many obscure cases of gall-bladder infection, for instance, being traceable 
to this organism. 

The presence of non-pathogenic organisms seems to be an essential for 

proper performance of intestinal function. This idea, orignally advanced by 

Pasteur, has been denied by Schottelius, Nuttall, and Thierfelder, although 

their experiments extended only over periods of 17 days. These intestinal 

^ See MacNeal and Chace, Arch. Int. Med., 1913, XII, 178; also, MacNeal, Am. Jour 
Med. Sc, 1913, CXLV, 801; Rahe, Jour. Infect. Dis., 1Q15, XVI, 210. 
2 See Mattill and Hawk, Jour. Exper. Med., 191 1, XIV, 433. 



THE FECES 



129 



bacteria not only aid digestion, but also prevent a certain amount of abnormal 
decomposition, owing to the fact that they inhibit the development of foreign 
types to a large extent, especially under normal conditions. These normal 
bacteria may under certain conditions, however, become pathogenic, but only 
when the intestinal wall loses its continuity. In case the normal bacteria of 
the intestine become too numerous, the products formed by their activity upon 
protein material may be absorbed and bring about certain toxic ejEfects.^ It 
is interesting in this connection to find that the bacteria are diminished in 
amount in chronic constipation, according to Strasburger. The toxic 
symptoms must, therefore, be referable to the absorption of other than bac- 
terial products of decomposition, or the products have increased toxicity. 
For the best conditions to exist the symbiotic relations of the intestinal bac- 
teria should be such that there are neither too few nor too many bacteria. 




Fig. 



-Cholera spirilla. {Pitfield.) 



The normal adult stool shows a preponderance of Gram-negative organ- 
isms (colon bacilli). Pathologically an increase in this type may be noted 
(cholera spirilla) or Gram-positive organisms may prevail. This Gram- 
staining variation in the bacteria of the feces is, also, largely influenced by 
changes in diet, bacteria of the Bacillus acidophilus and bifudus types be- 
coming quite prominent under a substitution of a low protein for a high 
protein diet. 2 

{a). The Cholera Spirillum. 

This organism, known as the comma bacillus, is about 2 microns long 
and 3^-2 micron thick. It is very actively motile and has a single delicate 
flagellum at one end. It stains easily with the ordinary bacterial stains and is 

^ See Zuntz, Die Naturwissenschaften, 1913, I, 7. 

2 See Herter and Kendall, Jour. Biol. Chem., 1910; VII, 203; Rettger and Horton, Cen- 
tralbl. f. BakterioL, Orig., i9i4,LXXIII, 362; Osborne and Mendel, Jour. Biol. Chem., 1914, 
XVIII, 177; Logan, Jour. Path, and BacterioL, 1914, XVIII, 527; Blatherwick and Hawk, 
Jour. Am. Chem. Soc, 1914, XXXVI, 147; Rettger, Jour. Exper. Med., 1915, XXI, 365; 
Torrey, Jour. Infect. Dis., 1915, XVI, 72; Logan, Lancet, 1916, II, 824; Nissle, Deutsch. 
Med. Wchnschr., 1916, XI.II, 1181; Hull and Rettger, Jour., BacterioL, 191 7, II, 47; 
Sisson, Am. Jour. Dis. Child., 1917, XIII, 117; Porter, Morris and Meyer, Ibid., 1919, 
XVIII, 254; Morris, Porter and Meyer, Jour. Infect. Dis., 1919, XXV, 349; Torrey, Jour. 
Med. Res., 1919, XXXIX, 415; Mendel, Am. J. Med. Sc, 1919, CLVTII, 297; Distaso and 
Sugden, Biochem. Jour., 1919, XIII, 153. 
9 



I30 



DIAGNOSTIC METHODS 



decolorized by Gram's method. The cultural peculiarities of this spirillum 
will be discussed in the last section of this book. This organism is usually 
recognizable in the stools of Asiatic cholera, which are the characteristic "rice- 
water'^ stools. The blood serum of patients affected with cholera will show 
very characteristic agglutination of cultures of these organisms. The blood 
is usually used in a dilution of i to 1 5, the reaction being observed in from 5 to 
20 minutes. 

Closely related to this comma bacillus of Koch is the bacillus of Finkler- 
Prior. This latter organism may be distinguished from the spirillum of 
Asiatic cholera by its morphology, the organism being larger and thicker than 
the comma bacillus, and by the appearance of the stab cultures on gelatin. 
The cholera spirillum forms a typical funnel-shaped depression^ while the 
bacillus of Finkler-Prior shows a stocking-like depression. The Finkler- 
Prior bacillus is found in cases of cholera nostras. It may be necessary for the 
absolute identification of these organisms to apply bacteriolytic tests with 
serum of animals immunized against a specific type of organism.^ (See 
Chapter XL) 




Fig. 23. — Bacillus typhosus, stained to show flagella. 
{Oertel after Frankel and Pfeiffer.) 

(b) Typhoid Bacillus. 

This organism, discovered by Eberth, is so similar in morphology to 
numerous other organisms, especially to the Bacillus coli communis, that 
simple staining methods do not suffice for its detection. The characteristic 
stool of typhoid fever is a copious watery stool, having a strong odor and 
an alkaline reaction. This stool is known as the " pea-soup^' stool, and may 
be tinged with blood and contain many pus-cells. 

The typhoid bacilli are medium-sized organisms with rounded ends, 
generally short, but sometimes long or thread-like and frequently showing 
faintly stained, sharply defined areas in their protoplasm. They are actively 
motile and have both polar and lateral flagella. This organism stains with 
the ordinary dyes and is decolorized by Gram's method. 

1 See Public Health Reports, 191 2, XXVII, 371. 



I 



THE FECES 13I 

Method of Drigalski and Concradi. ^ 

Three pounds of minced beef are mixed with 2 Hters of water and allowed 
to stand overnight. The beef is then pressed and the juice boiled for one 
hour and filtered. To the filtrate are added 20 grams of Witte's peptone, 
20 grams of nutrose, and 10 grams of sodium chlorid. Boil this mixture one 
hour and filter. To the filtrate 60 grams of agar are added and the mixture 
boiled for three hours, one of which should be in the autoclave. Slightly 
alkalinize the mixture to litmus-paper, filter, and boil for one-half hour. To 
this hot agar solution, which should be now about 60°, add the following litmus- 
lactose solution. Two hundred and sixty c.c. of litmus solution are boiled for 
10 minutes, after which 30 grams of chemically pure lactose are added and 
the mixture boiled for 15 minutes longer. This . litmus-lactose solution is 
added while boiling to the hot agar solution, the mixture being well shaken 
and again faintly alkalinized to litmus. Four c.c. of a hot sterile 10 per cent, 
solution of sodium carbonate and 20 c.c. of a freshly prepared o.i per cent, 
solution of crystal- violet B. (Hochst) in warm sterile distilled water are then 
mixed in. This medium may be poured directly into plates or kept in 
flasks. It soon hardens to a firm mass and does not become dry readily. 

The principle upon which the use of this medium depends is that in 
the presence of both lactose and protein the colon bacillus will first attack 
the milk-sugar, while the typhoid bacillus will act upon the protein. In the 
presence of litmus the colonies of colon bacilli become distinctly red, while 
those of the typhoid bacillus are blue. The crystal-violet inhibits the 
growth of many of the other organisms, especially of the acid-producing type." 

If the stool be fluid, as is usually the case, one series of two plates is inocu- 
lated with the undiluted stool, another with a stool diluted with 10 volumes 
of sterile normal salt solution, while other dilutions, such as i to 100 and i to 
1000 may also be made. If the stool is solid it is rubbed up into a homogene- 
ous mass with sterile salt solution and the various dilutions made as above. 
In making the inoculations from the stools, the material is rubbed over the 
surface of the medium, the plates being left open to allow the surface to dry. 
The dry plates are then placed in the incubator at 37° and examined at the 
end of 24 hours. Any contamination of the media will be killed by the crys- 
tal-violet. 

It frequently happens that certain strains of the paratyphoid bacillus 

1 Ztschr. f. Hyg. u. Infektionskrankh., 1902, XXXIX, 283. For a discussion of the use 
of benzin in differentiating members of this group see Benians, Ztschr. f. Chemotherap., 
1913, II, 28; Bierast, Centralbl. f. Bakteriol, I. Abt., Orig., 1914, LXXIV, 348; Schmitz, 
Munch, med. Wchnschr., 1914, LXI, 2115; Jaffe, Wien. klin. Wchnschr., 1915, XXVIII, 418. 
Hall, Berl. klin. Wchnschr., 1915, LII, 1326. 

2 Quite an extensive amount of research has centered about the use of brilliant green in 
culture media used for isolation of typhoid bacilli. This dye seems to afford an excellent 
enrichment medium. See Krumwiede, Pratt, and McWilliams, Jour. Infect. Dis., 1916, 
XVIII, i; Teague and Clurman, Ibid., 647 and 653; Robinson and Rettger, Jour. Med. 
Res., 1916, XXXIV, 363; Teague and Clurman, Ibid., 1916, XXXV, 107; Meyer and 
Stickel, Jour. Infect. Dis., 1918, XXIII, 48; Krumwiede, Kohn, Kuttner, and Schumm, 
Ibid., 275. For other methods in connection with the isolation of this organism see Kem- 
per, Jour. Infect. Dis., 1916, XVIII, 209; Tonney, Caldwell, and Griffen, Ibid., 239; Holt, 
Harris, and Teague, Ibid., 596; Ecker, Ibid., 1918, XXII, 95; Hulton and Frankel, Ibid., 
191 8, XXIII, 380; Kligler, Jour. Exper. Med., 191 8, XXVIII, 319; Pacini and Russell, 
Jour. Biol. Chem., 1918, XXXIV, 43. 



132 DIAGNOSTIC METHODS 

develop blue colonies in this medium. It is, therefore, necessary for absolute 
differentiation that the agglutination test described under blood be carried 
out. In this way the typhoid bacillus may be absolutely identified, especially 
in the presence of its well-known morphological characteristics.^ 
Method of Kendall and Day. 
This method- permits of rapid diagnosis and is especially of importance 
in the examination of stools of suspected "carriers." The culture medium is 
a modification of that of Endo. Plain nutrient sugar-free agar is prepared as 
follows: 1000 c.c. of cold tap water, 15 grams of powdered agar, 10 grams of 
Witte's peptone, and 3 grams of Liebig's extract of beef are mixed and cooked 
in the double boiler for one hour. Just alkalinize to litmus by careful 
addition of sodium hydrate solution, cook for 15 minutes and filter through 
absorbent cotton. This filtrate is then stored in flasks containing known 
amounts (100 c.c.) and is sterilized in the autoclave. When it is desired to 
use this media, add i gram of chemically pure lactose to each 100 c.c. and heat 
in the sterilizer until the media is melted and the lactose thoroughly distri- 
buted. Now prepare a mixture of i c.c. of a filtered 10 per cent, alcoholic 
solution of fuchsin and 10 c.c. of afresh 10 per cent, aqueous solution of sodium 
sulphite. 2 Add i.c.c of this sterilized and decolorized fuchsin mixture to 
each 100 c.c. of the liquid lactose media and mix thoroughly. Plates are 
poured and allowed to harden (with the covers removed) in the incubator for 
30 minutes, when they are ready for inoculation. 

A small portion of feces is emulsified in 10 c.c. of sugar-free^ bouillon 
and incubated for i hour at 37°C. This supension is rubbed gently but 
firmly over the surface of the agar plates by means of sterile hooked glass 
rods and incubated for 18 hours at 37°C. The translucent, colorless, "dew- 
drop" colonies are removed to small tubes, which contain i c.c. of sugar-free 
bouillon and have been kept at 37°C. prior to use.^ Incubate for 2 hours and 
make agglutination tests, using a known serum of high agglutinating power 
(see blood). 

(c) The Bacillus of Dysentery. 

Bacillary dysentery is distinctly different from the amebic type of dysen- 
tery. The bacillus dysenteriae, or Shiga's bacillus, is now general y recog- 
nized as the specific organism of this type of dysentery. 

Flexner found a similar bacillus in the dysentery of the Philippines, while 
Kruse has found practically the same type in Germany. In the United 

^ For a discussion of the differentiation and characteristics of Paratyphoid bacillus A 
and B, see Bacteriology of Blood. 

2 Jour. Med. Research, 1911, XXV, 95. See, also, Schmidt, Deutsch. med. Wchnschr., 
1915, XLI, :^:^; Hirschbruck and Diehl, Ibid., 606. 

•' If preferred Andradi's indicator (a 0.5 per cent, aqueous solution of acid fuchsin de- 
colorized by the careful addition of 10 per cent, sodium hydrate solution) may be sub- 
stituted. See Kahn, Jour. Bacterid., 1918, III, 547, 

4 This sugar-free broth is specially prepared to remove the muscle-sugar. Meat extract 
is inoculated with colon bacilli and incubated for 16 hours. Boil and strain through cloth. 
The filtrate is then made up as ordinary bouillon. 

■^ Or the colonies may be transferred to Russell's medium (litmus-lactose-glucose agar) as 
recommended by Lumsden fPub. Health Reports, 191 2, XXVII, 789). See also Kendall 
and Ryan, Jour. Infect. Dis., 1919, XXIV, 400, for a double sugar medium of saccharose 
(i per cent.) — mannitol (o.i per cent.) agar. 



THE FECES 1 33 

States Flexner and Harris find an organism which answers the description of 
the ordinary Shiga bacillus. The variation between the different organisms 
described is very slight, so that one may generally consider them as varieties 
of the same species, although certain cultural and agglutinating differences 
are observed. 

The Shiga bacillus is a short rod with rounded ends, much resembling the 
typhoid bacillus but much plumper. It does not seem to have very active 
motility as far as progression is concerned, although it does show a high degree 
of molecular motility. It stains with the usual dyes and is decolorized by 
Gram's method. The only sure method of identification of the various types 
of the dysentery bacillus seems to be the agglutination test. 

The fecal material is best obtained by curettage of the rectum, as these 
bacilli seem to be present in the mucus and are thus more easily concentrated. 
The cultural peculiarities of this organism will be found in a later section. 
{d) The Tubercle Bacillus. 

The examination of the stools for tubercle bacilli is not always satis- 
factory. The enormous number of bacteria of the feces may prevent a recog- 
nition of the tubercle bacillus even though it is present. In the feces we find 
certain organisms which are acid-fast, such as the timothy bacillus, which is 
very closely related in morphology and staining characteristics to the tubercle 
bacillus. We should be on our guard, therefore, lest we make a wrong 
diagnosis. 

If these organisms are present on repeated examination and there are 
clinical symptoms pointing to such a trouble of the bowel, one may give a 
presumptive diagnosis of tuberculosis, remembering that the tubercle bacilli 
may have come from swallowed tubercular sputum.^ 

In selecting the material for examination, pick out the particles of mucus, 
especially those which are blood-stained or purulent. One may be much 
more certain of definite results if the antiformin or Petroff method be used 
upon the feces, which has been rubbed up with distilled water (see p. 18). 
The staining methods are as usual. 

The Bacillus aerogenes capsulatus (Bacterium Welchii; gas bacillus) is 
fast coming into prominence as an etiologic factor in many cases of intestinal 
disturbance associated with diarrhea, although it is possibly a normal habi- 
tant of the intestinal tract of children. ^ 

VI. Parasitology of the Feces 

In the examination of the feces for parasites one should obtain the feces 
as fresh as possible. This is especially the case where the examination for 
protozoa such as the ameba is to be made. The feces should be kept in a 
warm vessel prior to the examination, as the motility of these unicellular 

^ See Laird, Kite and Stewart, Jour, Med. Research, 1913, XXIX, 31 ; Bergstrand, Hygiea, 
1915, LXXVII, 97; Petroff, Jour. Exper. Med., 1915, XXI, 38; Keller and Moravek, Med. 
Record, 1915, LXXXVIII, 864; Leffler, Hygiea, 1916, LXXVIII, 1809. 

2 See Kendall and Smith, Boston Med. and Surg, Jour., 1911, CLXIV, 306; Kendall, 
Ibid., 1912, CLXV, 75;Orton, Jour. Med. Research, 1913, XXIX, 287; Knox and Ford, Bull. 
Johns Hopkins Hosp., 1915, XXVI, 27; Morse, Am. Jour. Med. Sc, 1915, CXLIX, 17. 



134 DIAGNOSTIC METHODS 

organisms is shown only with great difficulty after they have beco'me cold. 
The other types of protozoa are less sensitive to changes in temperature and 
show their active motihty, providing the feces be examined sooxi after voiding. 
It seems to be generally accepted that these organisms are the more easily 
found the more fluid, more mucoid, and more alkaline the feces. The par- 
ticles for examination should be preferably the masses of mucus which can be 
found by careful search in the liquid stool. The organisms are best examined 
in their fresh condition, as the staining agents usually require fixation of the 
specimen and consequent death of the parasite.^ 

In the examination of the feces for parasitic ova the following method of 
Yaoita (Deutsch. med. Wchnschr., 191 2, XXXVIII, 1540) is, in my opinion, 
the best. From several different parts of the fecal mass take portions the 
size of a pea. Place in a test-tube and add 5 c.c. of 25 per cent, antiformin 
solution. Mix thoroughly and warm but do not boil. Allow to cool, add 
5 c.c. of ether and shake well. Filter the mixture through one layer of gauze 
and cetrifuge the filtrate for one minute. Four layers are formed, in the 
lower of which are the ova along with the resistant cellulose, connective tissue 
and muscle fibers and salts. Pour off the supernatant fluid and treat the sedi- 
ment with dilute HCl and a little ether. Shake well and centrifuge again. 
This latter process is repeated two or three times. The ova are thus freed 
from most of the other fecal material and may be detected readily by micro- 
scopic examination of the sediment.^ 

(i) Protozoa. 

The protozoa are unicellular animal organisms. These, although living 
occasionally symbiotically, are more usually found as isolated single organ- 
isms. A few of these organisms are sufficiently large to be detected by the 
naked eye, but the majority are minute and require the finer microscopic 
detection. They consist essentially of a mass of protoplasm (cytoplasm or 
sarcode), with differentation for functional purposes (organelles) of a variable 
character, constancy, and prominence. In the ameba, for instance, the 
sarcode may be separable into an internal distinctly granular portion known 
as the endosarc, and a peripheral clearer portion known as the ectosarc; a 
cell-membrane in some instances is a well-marked feature, while in others it 
is absent; and in some of the free-hving protozoa special external coverings of 
chitinous, siliceous, or chalky composition enclose the protozoon. Of the 
various parts the nucleus is, after the cytoplasm, the most constant, varying 
much in appearance, shape, size and number in the individual form (single 
nucleus of variable size and shape; double or dimorphic nucleus, a macronu- 
cleus of vegetal character, a micronucleus with creative function; polymor- 
phous nucleus, multiple nuclear granules more or less widely distributed in 
the cytoplasm). 

1 Ross (Further Researches into Induced Cell-reproduction and Cancer, Philadelphia, 
191 1 and 191 2) has advocated the method of embedding the suspected feces in agar-jelly 
which may be impregnated with the stain. See, also, Smithies, Arch. Int. Med., 19 12, IX, 
736. 

2 See Fauntleroy and Hayden, U. S. Naval Med. Bull., 1915, IX, 81; Hall, Jour. Lab. 
and Clin. Med., 191 7, II, 347; Bak, Nederland, Tjdschr. voor Geneesk., 191 7, II, 1117. 



THE FECES I$$ 

Not uncommon examples of specialization are met in the contractile 
vacuoles, in pigment spots, in mouth-like ingestion foci and their pits on the 
surface of many forms with relatively firm cell membrane, in the anus-like 
excretory points of the same form, or the peripheral motor organelles, in the 
sucking tubes of the suctoria, and in the hook-like fixation apparatus of the 
gregarines. 

Motile protozoa move in a variety of ways. The naked rhizopods move 
by a peculiar rolling due to currents in the internal substance of the cell or by 
the protrusion of the cell-substance as extensions or pseudopoda, these move- 
ments being always accompanied by change in the cellular shape of the 
animal. Ciliates and flagellates move through the activity of the special 
cuticular appendages known as ciliae and flagella. 

(a) Rhizopoda (Sarcodina). 
Amoebina. 

The classification of Amebae has been a more or less difficult task, owing 
to lack of knowledge of the complete life cycle of many of the so-called species; 
the simple morphology of these organisms; the difficulties inherent in the 
study of such delicate cells; and the conflicting opinions of protozoologists 
as to the data upon which generic and specific classification should be based 
(Craig). The classification of Calkins,^ although not entirely satisfactory 
appears to be the most logical. He divides the genus Ameba into seven gen- 
era as follows: Ameba, Vahlkampfia, Nagleria, Craigia, Trimastigameha, 
Entameha and Farameba. Only three of these, namely, Vahlkampfia, Craigia 
and Endameba, are of interest as parasitic amebae of man. The only 
species of the first of these three latter genera to be actually identified as 
parasitic in man is Vahlkampfia lobospinosa, first described by Musgrave 
and Clegg2 and later named by Craig. Of the second genus, two species 
have been shown to be parasitic, namely, Craigia hominis {parameba hominis) 
and Craigia migrans.^ Of the genus Endameba, no less than twenty-six 
distinct species have been described as parasitic in man, but many of these 
have been determined to be identical with some of those previously described. 
In this number, only three of the species of endamebse are of great interest 
to us, namely, Endameba histolytica, Endameba coli, and Endamebae gingivalis- 
A further type, which is non-pathogenic, has been described by Weyon 
and O'Connor under^ the name Endameba nana and seems to be a very 
common intestinal ameba among the troops. 

(a) Amoeba coli (endamceba histolytica). 

The ameba was first discovered in the large intestine by Lambl, although 

.we are indebted to Losch for the first accurate description of this organism. 

^ Tr. 15th. Internat. Cong. Hyg. and Demog., 191 2, II, 287; See, also, Craig, Jour. Med. 
Research, 1917, XXXV, 425. 

2 Bur. Gov't. Lab. Biol., Manila, 1904, No. 18; see, also, Craig, Arch. Int., Med-, 1914, 
XIII, 737. 

3 Craig, Am. Jour. Med. Sc, 1906, CXXXII, 214; Barlow, Am. Jour. Trop. Dis. and 
Prevent. Med., 1915, II, 680. 

* Jour. Royal Army Med. Corps, 191 7, XXVIII, i; Kofoid, Kornhauser, and Plate, 
Jour. A. M. A., 1919, LXXII, 1721; Kofoid, Kornhauser and Swezy, Arch. Int. Med., 1919, 
XXIV, 35. 



136 DIAGNOSTIC METHODS 

He did not believe it to be the cause of dysentery, but regarded it as a sec- 
ondary invader. Since that time much work has been done, a controversy hav- 
ing arisen regarding its specificity. In a great many cases of dysentery this 
organism is not found, but in its stead bacteria, especially the Shiga bacillus, 
are present in large numbers. This bacillary dysentery is something entirely 
different from the amebic type of this disease. It is true that in certain cases 
of dysentery as well as in normal individuals, amebae are found which are 
differentiated with great difficulty from the true amaeba coli. There seem to 
be two distinct types : one pathogenic, to which Councilman and Lafleur give 
the name amoeba dysenteriae, which Losch styles amoeba coli, and which 
Schaudinn designates endamoeba histolytica; a second type, which is non- 




FiG. 24. — Amoeba coli. (Hemmeter.) 

pathogenic, has been styled by the first writers amoeba coli Losch and by 
Schaudinn endamoeba coli. Viereck^ has described a form, which is patho- 
genic and is known as the endamoeba tetragena. This form is identical with 
the endamoeba histolytica. These forms have not been cultivated. The 
cultivated forms are non-pathogenic to man.^ 

One may sum up the points relative to the pathogenicity of the endomoeba 
histolytica as follows: (i) It appears in a form of dysentery which is anat- 
omically characterized by peculiar ulcerations which are markedly different 

1 Beiheft z. Arch. f. Schiffs. -u. Tropenhyg., 1907, I, i. 

2 See Craig, Arch. Int. Med., 1911, VII, 362; Am. Jour. Med. Sc, 1912, CXLV, 83; Jour. 
Med. Research, 191 2, XXVI, i; War Dept., Office of Surgeon General, Bull. 2, 1913, 95; 
Jour. Am. Med. Assn., 1913, LX, 1353; South. Med. Jour., 1913, VI, 370; Jour. Infect. Dis., 
1913, XIII, 30; Whitmore, Arch. Int. Med., 1912, IX, 515; Darling, Arch. Int. Med., 1913, 
XI, I and 495; Jour. Am. Med. Assn., 1913, LX, 1220; James, New York Med. Jour., 1913, 
XCVIII, 702; Couret and Walker, Jour. Exper. Med., 1913, XVIII, 252; Williams and 
Calkins, Jour. Med. Research, 1913, XXIX, 43; Giffin, Jour. Am. Med. Assn., 1913, LXI, 
675; Walker and Sellards, Editorial, Jour. Am. Med. Assn., 1914, 300; James, Ann. Trop. 
Med. and Parasitol., 1914, VIII, 133; Craig, Arch. Int. Med., 1914, XIII, 737 and 917; 
Sellards and Baetjer, Am. Jour. Trop. Dis. and Prev. Med., 19 14, II, 231; Bull. Johns Hop- 
kins Hosp., 191 5, XXVP, 45; Couret, Am. Jour. Trop. Dis. and Prev. Med., 1915, II, 450; 
Cutler, Jour. Path, and Bacteriol., 1918, XXII, 22; Parasitol, 1919, XI, 127. 



THE FECES 



137 



from the diphtheritic inflammatory processes of the bacillary dysentery. (2) 
The more recent the case the more numerous are these parasites; (3) they 
are deposited in the dysenteric ulcers and tend to pass into the deeper tissue 
appearing as true tissue parasites; (4) they frequently deposit themselves in 
the liver causing abscesses which contain these organisms in large numbers 
and practically no other infectious material; (5) by injection of amebae- 
containing feces into the large intestine of animals typical amebic dysentery 
may be caused. It is to be borne in mind that symbiosis with bacteria is 
apparently necessary for the development of amoebae. 

The endamoeba histolytica is an actively motile, roundish, pear-shaped, 
oval, or irregular unicellular organism having an endosarc which is typically 
granular and may contain lebcocytes, red blood-cells, bacteria, particles of 








Fig. 25. — Coccidium hominis, from intestine of rabbit: i, A degenerate epithelial cell 
containing two coccidia; 2, free coccidium from intestinal contents; 3, coccidium with four 
spores and residual substance; 4, an isolated spore; 5, spore showing the two falciform 
bodies — X 1140. (Tyson after Railliet.) 



food, or pigments which the parasite has ingested, and shows the clear hyalin 
ectosarc, which is, perhaps, best seen in the pseudopoda. The pseudopoda 
are the typical motile portions of the parasites, these projections being 
thrown out from any point of the periphery, the protoplasm seeming to flow 
into them and drawing the animal after it. The parasite may not move, but 
will ch?ange its external appearance by throwing out these pseudopoda in 
various directions. The nucleus is a homogeneous, little ref ractile, chromatin- 
poor, spherical mass, about 6 microns in diameter (the diameter of the organ- 
ism itself ranging between 10 and 50 microns, the average being 35). This 
nucleus is not always clearly visible, appearing much more frequently in the 
animal killed by corrosive sublimate. In the granular endosarc one fre- 



138 DIAGNOSTIC METHODS 

quently sees several vacuoles which may or may not pulsate, the general 
opinion being that pulsation is absent, although change in shape is frequent.^ 

As Craig^ states, "It is now well known that it is not the patient sick 
with acute symptoms of endamebic dysentery who is the greatest source of 
danger to the uninfected but the apparently healthy individual, or one 
who has recovered from the acute attack, but who is carrying cysts of Enda- 
meba histolytica in his stools. The Cystic stage of Endameba histolytica 
is the chief agent of infection in this disease, for while the vegetative motile 
forms may produce infection when ingested, the evidence is conclusive that 
in both man and animals the ingestion of cysts is followed by infection* in the 

vast majority of instances Cysts of Endameba histolytica occur 

in chronic infections; in cases in which treatment has not resulted in the 
destruction of all the endamebse; and in apparently healthy individuals who 
have never presented symptoms of dysentery. For the latter class the 
name 'contact carrier' is suitable, while for those individuals who develop 
cysts during convalescence from an acute attack of endamebic dysentery 
the name 'convalescent carrier' is used." 

On account of the great source of danger from these carriers of amebic 
infection and, also, to the fact that such carriers harbor these cysts for many 
months and possibly years, it is important that the stools be searched care- 
fully for these cysts in every suspected case. According to Kofoid, Korn- 
hauser and Swezy,^ the cysts of endameba histolytica are distinguished by 
their glassy, refractive, irregularly vacuolated or (in small cysts) almost 
homogeneous cytoplasm, with distinct nuclei with central granule and heavy 
rim never exceeding four in number, with relatively abundant mononu- 
clears and glycogen, when present, diffusely distributed. These cysts are 
usually spherical and fall into three general groups according to size, the 
largest being from 12 to 14 microns in diameter and the smallest from 7 to 
8 microns. The cyst wall is thin and rather easily penetrated by the Donald- 
son stain (mentioned below) and by hematoxylin stains. With the Donald- 
son stain the cytoplasm is first a bluish gray which changes to a yellow and 
then to a pink color, which gradually deepens to red. The cytoplasm is 
unevenly vacuolated and is very finely granular but the granules are not 
so evenly distributed as in Endameba coli. Chromatoid rods are found 
in a majority of the cysts. Nuclei one to four in number. The chromatin 
appears to be evenly distributed on the nuclear membrane, which seems to 

1 If one wishes to stain such preparations, the methodjof Darhng](Jour. Am. Med. Assn., 
191 2, LIX, 292, and Science, 1913, XXXVII, 58) is reliable. Stain with Wright's stain in 
the usual way and follow this with Giemsa's stain until the film has a purple cast. Then 
plunge this into 60 per cent, alcohol to which 10 to 20 drops of aqua ammonias have been 
added. Differentiate in^this way until the film has a violet color. Dry and examine. See, 
also, Craig, War Dept., Ofl&ce of Surgeon General, Bull, i, 1913, n; Donaldson, Lancet, 
1917, I, 571, advocates the use of a mixture of equal parts of (i) five per cent, aqueous 
solution of potassium iodid saturated with iodin, to which an equal volume of ether is 
added, and. (2) a saturated aqueous solution of rubin S or of eosin. A loopful of feces rnixed 
with a few loopsful of the above mixture is placed on a glass slide and covered with a 
clean cover-glass. The cysts stand out as brilliant yellow or greenish yellow spheres with 
a sharply defined outline. 

2 Mil. Surg., 1917, XI^, 286 and 423. 

3 Arch. Int. Med., 1919, XXIV, 35. 



THE FECES 139 

be thicker than in Endameba coli, and there is present a distinct central 
granule, visible under proper focus as a clearly defined central dot.^ 

The stools of this amebic dysentery are thin and watery, show an alkaline 
reaction, and have a peculiar lime-like odor. Much mucus, blood, and occa- 
sionally many pus-cells are found for which reason the mucus should be 
selected for examination in case these organisms are suspected.^ Frequently 
one may obtain better specimens for examination by examining mucus which 
is obtained with a rectal tube. 

{(3) Endamoeba coli (amoeba coli Losch). 

This parasite varies in size between 10 and 15 microns. The hyaline pro- 
toplasm of the pseudopoda is not distinctly differentiated from the ectoplasm. 
It is opaque, gray in color, and its nucleus is sharply defined, being character- 
ized by its richness in chromatin. The movements of this organism are not 
as rapid as those of the endamoeba histolytica, which does not show as active 
phagocytic power. According to Craig, about 65 per cent, of normal persons 
show these non-pathogenic endamoeba coli in the feces. 

Cysts of Endameba coli are generally spherical and range from 14 to 22 
microns in diameter. Cyst wall is thicker than that of the histolytica and 
is clearly defined. Rather easily attacked by the iodin-eosin stain of Donald- 
son but acts irregularly toward iron hematoxylin. Cytoplasm is more 
coarsely but more uniformly granular than that of the histolytica. Gly- 
cogen rarely present except in cysts having fewer than eight nuclei. Chro- 
matoid bodies rarely seen. Nuclei generally eight in number, but large 
cysts may contain sixteen. These appear clear and definite in the iodin-eosin 
stain. Each nucleus has a central granule and peripheral chromatin situated 
in the nuclear membrane in uneven masses, giving, at sharp focus, a rather 
ragged inner edge to the refractive ring of chromatin material.^ The nucleus 
of Endameba nana shows no central granule and the peripheral chromatin 
is massed in a single large clump at one point on the nuclear membrane. 

(b) Sporozoa. 
Coccidium hominis (coccidium perforans; cystospermium hominis). 

This organism appears in the feces as an oval or spherical parasite about 
22 microns long and showing a thin periphery. A large number of nuclei are 
usually observed! 

The infection with these organisms seems to arise from rabbits in whose 
intestines these parasites develop in large numbers. 

^ See Fischer, Deutsch. Arch. f. klin. Med., 1915, CXVIII, 129; Ravaut and Krol- 
unitsky, Presse Med., 1916, XXIV, 169 and 289; Sanford, Jour. A. M. A., 1916, LXVII, 
1923; Cropper and Rowe, Lancet, 1917, 1, 179; Boeck, Univ. Calif. Pub. ZooL, 1917, XVIII, 
145; Carles and Barthelmy, C. R., soc. biol. de Paris, i9i7,LXXX, 402; Mathis and Mer- 
cier, Presse Med., 191 7, XXV, 114; Dobell and Jepps, Brit. Med. Jour., 191 7, I, 607; 
Walker and Emrich, Jour. A. M. A., 1917, LXVIII, 1456; Ravaut, Presse Med., 1917, XXV, 
81; Wenyon and O'Connor, Jour. Royal Army Med. Corps, 1917, XXVIII, 686; Shimura, 
Jour. Exper. Med., 1918, XXVIII, 415; Mathews, Ann. Trop. Med. and Parasitol., 1918, 
XII, 17; Smith, Ibid., 27; Cort and McDonald, Jour. Infect. Dis., 1919, XXV, 501; Haig, 
Lancet, 1919, II, 823. 

2 Musgrave and Clegg advise the administration of a saline cathartic and the later ex- 
amination of the fluid portion of the stool. 

^ See Kofoid, Kornhauser and Swezy, loc. cil. 




I40 DIAGNOSTIC METHODS 

(c) Flagellates. 
(a) Trichomonas intestinalis. 

This organism was first studied by Marchand and Zunker and later 
elaborated by Grassi, Roos, and Janowski. It is probably identical with the 
one known as trichomonas vaginalis which may live in the 
vagina, the urethra, large and small intestine, the stomach, 
and may be found in the sputum. Various forms have been 
described as being found in the intestine, but they are in all 
probability the same organism; among these we find protoryxo- 
myces coprinarius, monocercomonas hominis, cim(Bnomonas 
hominis, trichomonas hominis, cercomonas coli hominis, and 
cercomonas seu Bodo urinarius. 

This is a colorless protozoon of a pyriform or spindle shape, 
rounded in front and bearing three flagella which are apt to be 
merged at the base and easily lost, the posterior end pointed 
but not bearing a flagellum. It is from 20 to 25 microns in 
Fig. 26.— leiigth and 8 to 12 broad. Along the body, starting from the 
Trichomonas base of the flagella, runs an undulating membrane in a some- 
(Tyson ) * what spiral manner to the posterior end. It has a finely 
granular cytoplasm and at its anterior end a vesicular nucleus, 
behind which one or more non-pulsating vacuoles may be seen. At times this 
organism may be observed to assume an ameboid form, the movements of 
the flagella having then ceased and projections resembling pseudopoda being 
observed.^ 

{^) Cercomonas hominis. 

This organism was first studied by Davine and has been found by many 
other workers. It is known under the names of cercomonas intestinalis, 
monocercomonas hominis, cimcenomonas hominis. The adult organism is a 
small, colorless, pyriform parasite, with round anterior end provided with 
one long flagellum and a pointed posterior end. It is 8 to 10 microns long 
and has no undulatory membrane, as has the trichomonas intestinalis. 

(7) Megastoma entericimi. 

This organism was first found by Lambl 'V^ ,^^^^ ^^^^^ 

in the feces of children. It is known under y^^ -<r^^^^^-^«>-. 
several names among which are Lamhlia in- Fig. 27. — Cercomonas hominis; 
testinalis, hexamitus duodenalis, dimorphous ff^^^/^^f ^""^ ^' '^^^^'' varieties. 
muris, and megastoma intestinale. This para- 
site is a colorless pear-shaped protozoon with a rounded anterior end and a 
pointed posterior end bearing a pair of flagella. The anterior end has one 
side concave with a raised border or lip, one pair of flagella, arising at the 
anterior border of this disk-like concavity, and two pairs together from its 
posterior margin. The cytoplasm is finely granular and the dumb-bell shaped 

1 See Castellani, Jour. Trop. Med. and Hyg., 1914, XVII, 65; Lynch, New York Med. 
Jour., 1915, CI, 886; Jour. Parasitol., 1916, III, 28; Rhamy and Metts, Jour. A. M. A., 1916, 
LXVI, 1 190; Chase and Tasker, Ibid., 191 7, LXVIII, 1528; Chalmers and Pekkola, Jour. 
Trop. Med., and Hyg. 1916, XIX, 142, report the finding of a new intestinal flagellate, 
octomitus hominis, with six anterior flagella. 



THE FECES 



141 



I 




I 



nucleus is anteriorly about the middle level of the concavity. Vacuoles are 
absent and sohd inclusions are never observed. The length of these organ- 
isms is from 15 to 16.5 microns, while the width is from 10 to 12.5 microns. 
The number of these organisms found in the feces may be very large. The 
surest points for their diagnosis seem to be the concavity and the dumb-bell 
shaped nucleus. The source of infection for man is the drinking of infected 
water. ^ 

{d) Infusoria. 

Balantidium coli (paramoecium 
coll) . This parasite is colorless, ovoid 
in shape, 70 to 100 microns long and 50 
to 70 microns broad, having a finely 
granular cytoplasm containing frag- 
ments taken from the intestinal ma- 
terial, and a clear ectoplasm showing 

numerous longitudinal striations. It Fig. 28.— Megastoma entericum, showing 

J 1^1 ^^^ ^-1 disk-surface and lateral views in larger 

IS covered completely with actively ^^^^^^^ ^^^ three epithehal cells with at- 

motile cilia which are more dense tached examples to the right. {Tyson.) 
about the funnel-shaped mouth 

which extends about one-fourth the length of the body. The nucleus is kid- 
ney shaped and is usually accompanied by one or more accessory nuclei, while 
two or more contractile vacuoles are seen which pulsate to a slight extent. 

This parasite has been found in connection with various types of diarrheal 
affection and also in persons entirely free from intestinal symptoms. It may 
be present in the stools in very large numbers, being found especially in the 
colon, but in severe cases also in the jejunum. The hog is the most common 
host of this parasite, which at times shows marked pathogenicity (see 
Bowman 2). 

(2) Entozoa (Enthelmintha). 
{A) Platodes (Flat Worms). 
(a) Cestodes (Tape-worms). 

The cestodes are naked, flat worms of elongated ribbon shape, endopara- 
sitic, at least in their adult stage, and in many instances in all stages, without a 
digestive canal, and always more or less distinctly divided into segments. The 
entire parasite, or strobile, may be looked upon as a colony of individuals 
united in ribbon fashion from their mode of origin, for convenience in their 
development and functional performance; the various segments being derived 
by a process of constriction from the originally acquired parasite, which is spo- 
ken of as the head, nurse, or scolex of the strobile. A characteristic of the ces- 
todes is the differentiation of two developmental stages: the first, cysticercus 

^ See Fantham and Porter, Brit. Med. Jour., 191 7, II, 139. 

-Jour. Am. Med. Assn., 1911, LVII, 1814. See, also, Hinkelmann (New York Med. 
Jour., 1915, CI, 200), who finds these parasites present in water supplies. The same 
author discusses the cultivation and reproduction of this parasite in N. Y. Med. Jour., 1919, 
■ CIX, 235. 




142 DIAGNOSTIC METHODS 

stage, in which the connective tissue or parenchymatous organs are invaded, 
and, secondly, the development of the sexually mature animal in the 
intestine. The scolex obtains entrance as a larva into the intestinal tract of 
the host, becomes attached by a special fixation apparatus to the mucous 
membrane, and there develops into the adult parasite, forming the anterior 
extremity of the strobile in the developed worm. The head is usually a very 
small and inconspicuous object of globular, pyriform or club shape with a 
short posterior extension, spoken of as the neck. In the middle of the frontal 
face of the head there is often a small prominence, known as the rostellum, 
about which may be arranged in one or more rows, as one of the means of 
fixation of the parasite, small booklets as a crown. As more constant means 
of fixation the head is provided with two or four 
suckers, rounded or linear depressions with more 
or less definite lips. Back of the head by a process 
of constriction from the neck, the segments, also 
known as links or proglottids, arise, the newest form 
always being placed between the neck and the next 
older Hnk. Thus the older segments are always 
separated more and more from the head by each 
newly formed proglottid, each as it grows older and 
recedes further from the neck developing in size; 
Fig. 29.— Balantidiumcoli: the length of the strobile being thus dependent upon 

a, Nucleus; 6, vacuoles; ^hg ^wo factors, growth of the individual length and 
c, cytostome, with pit and . 7 ^ , 

peristome; d, ingested the new formation of segments. These new seg- 
material. {Tyson after nients as they are first formed are usually very short 
and proportionately broader, but as they increase 
in size with age they generally enlarge especially in their long diameter, 
and come to be more or less square. The number of these links may vary 
from three or four to several thousands, the length of some worms being 100 or 
more feet. The structure of each link and hence of the whole strobile includes 
an interior or matrix of an indeterminate connective reticular material, from 
which the various organs appear to develop and in which they are imbedded; 
over which are to be recognized exteriorly a delicate cuticle and beneath the 
latter, two layers of so-called muscle, the outer layer longitudinal and the 
inner transverse and circular.^ 

Aside from the common parts the various links may be looked upon as in- 
dividuals. There is no digestive canal, all nutrition being obtained by the 
parasite from absorption of dissolved material from the fluids in the infested 
intestine. The only really highly organized parts are the generative organs, 
each link containing both male and female organs. The terminal links are 
the ones containing the most ova, while the links nearest the head are usually 
but partially developed. In their development the ova fill the canal of the 
oviduct more and more often causing the appearance of side pockets of more 
or less branching character. The terminal ripe links either actually contain- 
ing the ova or, after discharge of more or less of the original number, are apt to 

^ See Douthitt, 111. Biological Monographs, 1915, I, No. 3. 





THE FECES I43 

become separated from the strobile and be carried with the fecal matter from 
the intestine. Either with or without intermediate development the embryo 
is in some way, by water or in solids, carried into the alimentary canal of a 
second host. Arrived in this situation, both by 
its own activity and by passive convection by 
blood and lymph streams, the embryo penetrates 
the intestinal wall and becomes deposited in one 
or other situation as a larva, bladder-wortn, or 
cysticercus. This larval or cysticercus form is 
surrounded by a delicate connective tissue outer 
wall, derived from the host by a process of reac- 
tive inflammation, within which lies the true 
bladder-worm. This is essentially the head of 

the future parasite. This cysticercus, after re- '1 17 , , , , 

^ , . . . Fig. 30. — Head and neck, and 

mammg a variable period m the tissues, is de- ovum X300, of taenia solium. 

voured with the flesh of its host by a third Embryophore surrounded by 

. -^ vitellus. {Tyson after Gould.) 

(deiinitive) host. 

{a) Taeniidse. 

(i) Taenia solium. 

This parasite, which is derived from infection through the cysticercus 
cellulosae of pork, has been also named tcenia cucurbitina, taenia dentata, 
cystotcenia solium, and pork tape-worm. The average length of the strobile 
is 2 to 3 meters, occasionally reaching twice this measurement. The head 
is somewhat spherical or sHghtly tetragonal from the four rather prominent 
cup-like suckers with thick lips. The head varies from J^ to i mm. in di- 
ameter, while the suckers range from }4, to J^ mm. in diameter. It is pro- 
vided with a short, thick, rostellum bearing a double crown of booklets, usu- 
ally 28 in number. The neck is thin, about 3 cm. in length and is unsegmented. 
The proglottids number between 800 and 900, the fully grown and ripe seg- 
ments measuring from 9 to 12 mm. long and 5 to 6 mm. broad. The uterus 
consists of a large, median, longitudinal trunk with from 7 to 10 coarsely den- 
dritic branches on each side. The ova are round or oval, the shell very thin 
but surrounded by an embryonic layer which is thick and shows distinct 
radiating lines. These eggs are usually of a brownish color and may show 
on their interior the booklets of the embryo. 

This parasite in its adult stage is practically limited to the small intestine 
of man, while the larval form has been found in swine, monkeys, dogs, etc. 
It has been shown that careful cooking and prolonged and thorough salting 
and drying of the meat will destroy the vitality of the cysticercus cellulosae. 
It is, therefore, plain that any meat should be more or less thoroughly 
cooked before being eaten. 
(2) Taenia saginata. 

This parasite is the beef tape-worm and is also known as the tcenia medio- 
canellata, tcenia inermis, and tcenia dentata. The adult worm varies from 3 
to 8 meters in length, has a head from i to 2 mm. in diameter, tetragonal in 
shape without booklets or rostellum, with four cup-shaped suckfers each 0.8 



144 



DIAGNOSTIC METHODS 



mm. in diameter and placed at the corner of the frontal face. The ripe seg- 
ments are from 1 8 to 20 mm. long and 5 to 7 mm. broad. The uterus shows 
a distinct median longitudinal trunk with 20 to 35 lateral single or dichoto- 
mously branching and slender diverticula. The eggs are spherical with a 
thin shell surrounded by a thick radially striated embryonic shell. These 
eggs are from 30 to 40 microns long by 20 to 30 microns wide. 
(3) Taenia cucumerina. 
Infection with this parasite is relatively rare in the United States. It is 
found almost exclusively in children, the infection occurring through dogs and 
cats, the larval form of the parasites being found in 
the body lice and fleas. ^ This organism has other 
synonyms, among them being tcenia canina, tcenia 
moniliformis, tcenia elliplica, dipylidium 
caninum, and dipylidium cucumerinum. 
The parasite is from 15 to 35 cm. in 
length. The head is small, rhomboidal, 
with a clavate rostellum surrounded by 
three or four crowns of hooklets (48 to 60 
in number), suckers rather large with 
radially marked borders, and neck very 
short. The segments are from 80 to 150 
in number, the older ones being 8 to 11 

mm. in length and i to 3 mm. in breadth 

and often showing a reddish-brown color. T^nia cu- 
The links frequently swell out in the Ix^on^'^aUer 
middle so that the parasite has an ap- Leuckart.) 
pearance not unlike a chain of beads. 
A single uterus is common to the two oviducts,. consisting of a network of tubes 
in which the ova lie in groups, filling small saccules, each containing 10 or 15 
ova and surrounded by a reddish material which gives the color to the worm. 
The ova are spherical, 43 to 50 microns in diameter, and have a double wall. 
Within the wall one observes an embryo armed with hooklets. 
(4). Taenia nana. 
This worm is known as the " dwarf tape-worm ^^ and is perhaps best known 
in Italy and Southern Europe, although it has been found in many cases in the 





Fig. 31. — Head and 
neck of taenia saginata: A, 
retracted; B, extended. 
{Tyson after Gould.) 




Fig. 33. — Taenia nana:Xio. (Tyson after Gould.) 

eastern and southern portions of the United States.^ It has been called the 
tcenia cegyptica, hymenolepis nana, hymenolepis murina, and diplacanthus nana. 

1 SeeLins, Wien, klin. Wchnschr.. 191 1, XXIV, 1595; Ancona, Rif. Med. ,1916, XXXII, 
652. 

2 See Greil, Am. Jour. Dis. Child., 1915, X, 363. 



THE FECES 



145 



The infection with the ova is probably through the use of unfiltered water 
tainted with human or murine dejecta. It is most frequently seen in children, 
and inhabits the ileum, usually from the middle toward the ileocecal valve. 

The parasite is from 10 to 15 mm. in length and from 
0.5 to 0.7 mm. broad, is provided with a subglobular 
head measuring 0.2 to 0.3 mm. in transverse diameter, 
shows four large rounded suckers and a large rostellum 
retractile into an infundibulum. The rostellum is sur- 
rounded by a single row of characteristic hooklets, 24 to 
30 in number and 14 to 18 microns in length. The neck 
is rather long and slender, being followed by about 150 
small proglottids, which are broader than long (0.4 to 
0.9 mm. broad by 0.14 to 0.3 long). 

The ova are characteristic. They 
are round or oval in shape, 32 to 36 
by 42 to 56 microns in size, and have 
two distinct membranes. At each 
pole of the inner membrane is seen a 
small protuberance from which springs 

Fig. 34. — Head a number of clear refractile threads, 

and neck of taenia , • 1 j-i.-T_i.j- • Fig. t.<. — Ovum 

diminuta. {Tyson whlch are distributed m a waving ^^ t^nia diminuta. 

after Braun.) fashion through the substance inter- {Tyson after Braun.) 

mediate to the outer and inner walls. 





(5). Taenia diminuta. 

Synonyms. — Hymenolepis diminuta; hymenolepis flavo punctata; tcenia 
leptocephala; taenia jlavopunctata; tcEnia minima; and tcEuia varerina. 

The parasite is 10 to 60 mm. long, head small, globular, with four globose 
suckers situated close to apex, rostellum small, pyriform, and devoid of 
hooklets. The segments number 800 to 1300 and are broader than long. 
Gravid uterus nearly fills the segments, showing as transverse line when not 
ripe. 

The ova are round or slightly oval, yellowish in color, double-walled, 
inner wall showing shght protuberances at poles, a layer of albuminous 
material being seen between the walls. 



(6). Taenia echinococcus. 

This parasite in its adult stage is met with in the upper part of the small 
intestine of dogs, wolves, and jackals. The larval form, known as the hydatid 
cyst, is found in man, although more frequently in the ox, hog, horse, dog, 
cat, rabbit, etc. This disease is rare in America, and is acquired through 
association with the dog more frequently than in other ways. The most 
common seat of hydatid disease is in the liver. ^ 

The parasite is 2 to 5 mm. long, has a small subglobular head measuring 

^ See Phillips (Jour. Am. Med. Assn., 1913, LXI, 1981), who reports such a cyst in the 
pancreas. Delgado, Cronica Medica, 1916, XXXIII, 341; Llambias, Semana Medica, 
1916, XXIII, 359- 



146 



DIAGNOSTIC METHODS 



0.3 mm. in transverse diameter and bearing a rostellum with a double row of 
very characteristic hooklets (28 to 50 in number) and four prominent cup- 
shaped suckers. The neck is short and rather thick; proglottids three or four 
in number, the last of which is usually longer than the rest of the worm put 
together. Uterus consists of a thick longitudinal median trunk with a few 
short lateral branches. 

Ova spheroidal with thin radially striated shells and containing a granular 
hexacanthus embryo. Length of ova 30 to 60 microns, transverse diameter 
25 to 30 microns. 



(^ 




Fig. 36. — Taenia echinococcus: 
-a, Adult; b, head from echinococ- 
cus cyst. On left a detached hook- 
let, as seen in fluid from cyst. ( Ty- 
son after Coplin and Bevan.) 



Fig. 37. — Hydatid cyst, showing daughter cysts. 
In the lower part of field is a whitish mass containing 
parts of the walls of ruptured daughter cysts. The 
thick wall of the mother cyst is well shown. From 
liver of man, X %. {Coplin.) 



{b). Bothriocephaloidea. 
(i). Bothriocephalus latus. 

Synonyms. — Dibothriocephalus latus; tcenia lata; dibothrium latum; 
bothriocephalus latissimus; fish tape-worm. 

This parasite is most commonly met in the human intestine, but may be 
found in dogs and cats. It is most common in central Europe and in the 
maritime countries of Europe, British Islands, and Japan. The examples 
found in America occur in foreigners, as a rule. The ova, which are usually 
in large numbers in the feces, require for their future development immersion 
in water. The liberated embryo is then taken up by freshwater fish and con- 
veyed to man. 

Strobile 2 to 10 meters (20 in a few cases) in length, marked in ripe seg- 
ments by brownish central rosette (uterus with ova). Head elongated, 
almond shaped, 2 to 5 mm. long and 0.7 mm. transversely, with two lateral 



THE FECES 



147 



grooves or bothridia as suckers. Neck variable according to degree of con- 
traction. The segments number 3,000 to 4,000 and begin about 50 cm. from 
the head. The anterior hnks are poorly defined, in their growth increasing 
slowly in length but markedly in breadth. The ripe links measure 2 to 4 
mm. in length and 10 to 12 in width, with opaque brownish rosettes in the 
middle line. Uterus formed of a number of plicated tubes in the form of a 
rosette. 

The ova are brownish in color, ellipsoidal in shape, 68 to 71 microns in 
length and 44 to 45 in transverse diameter, have a thin shell, and a lid 

which may be opened or closed. 

The contents of the ova are 

coarsely granular or mulberry- 

like.i 

Infection with this worm is not 

always single, as high as 100 

worms having been reported in 

the same individual. Many cases 

of infection with this parasite are 

associated with a high-grade 

anemia, which is distinguishable 

from pernicious anemia only by the 

effects of removal of the parasite. 

(2) Dibothriocephalus cor- 
datus. 

This is a tape-worm of the 
same genus as the above and is 
parasitic in seals, being trans- 
mitted from them to man. It 
varies in length from 80 to 115 
-Bothrio- cm. Proglottids about 600, 7 to 8 
( yson ^^^ broad. The head is heart- 
shaped, 2 mm. long and broad, ^euckart.) 
The ova are similar to those of the latus, but are a little larger in size 




Fig. 38. 
cephalus latus. 
after Leuckart.) 



Fig. 39. — Diboth- 
riocephalus cordatus : 
adult. {Tyson after 



(3) Bothriocephalus sp. Ijima et Kuri-moto. 

Synonyms. — Diplogonoporus grandis; Krabbea grandis. 

Strobile measures up to 10 meters in length. Proglottids short and 
broad, head, neck, and number of segments unknown. Uterus rosette- 
shaped with several loops on each side. Ova brownish, operculated, oval, 
63 microns in length and 48 to 50 in width. Intermediate host unknown, 
probably fish. 

{^) Trematodes (Fluke -worms). 

The various forms of distoma, which belong to this class, are more prop- 
erly hepatic parasites, although they and their ova may at times appear 

^ See Rubenstone, N. Y. Med. Jour., 1916, CIV, 599; Magath, Jour. A. M. A., 1919, 
LXXIII, 85; Riley, Ibid., 11 86. 



148 



DIAGNOSTIC METHODS 



in the intestines and feces. A detailed discus- 
sion of these parasites will be taken up in the 
chapter on Parasites. 

(5) Nematodes (Roundworms). 

The nematode worms are unsegmented, 
elongate, circular or nearly so in their transverse 
section, cylindrical or more or less delicately fusi- 
form and tapering toward each end. They are 
with but few exceptions parasitic and include 
many important examples, which are parasitic in 
man. Intermediate hosts, so essential for the in- 
termediate development of the flukes and tape- 
worms, are practically absent in the nematodes. 



(a) Ascaridae. 

(i) Ascaris lumbricoides. 

This is the common round worm or maw- 
worm seen so frequently in children. The num- 
ber in a single host is usually small, but may be 
very large. Its habitat is the small intestine, 
but the eggs may occur in the vomitus as well as 
in the feces. 

The male worm is whitish to reddish-yellow 
in color; 15 to 17 cm. long, 3 to 3.5 mm. thick; 
elongate, fusiform; cuticle finely ringed; oral 
orifice terminal with three lips (one dorsal and 
the other two meeting in the median ventral 
line), each with fine denticulations on margins; 
at base of superior lip two papillae, one only at 
base of other two lips; posterior end terminating 
conically, curved ventrally, with two sligthly 
curved, short, equal spicules projecting from the 
subventral cloaca; 70 to 75 papillae on the 
ventral face of posterior end, of which seven 
pairs are postanal. 

The female parasite is 20 to 25 cm. long, 5 to 

5.5 mm. thick; anterior end and general appear- 

FiG. 40.— Ascaris lumbri- ^nce as in male; posterior end tapering, ending in 
coides: to left, male in lateral ., -^i^-i^^-i i 1.1 ir 

aspect; to right, female, ventral a COnical, pomted, straight tail, vulva at level of 

aspect, natural size. (Tyson f^j-gt third of body length (in a shghtly depressed 
after Railliet.) 1 t_ j\ U4- • i 

annular band) ; anus subtermmal. 

The ova are elliptical in shape, 50 to 75 microns long and 40 to 58 mi- 
crons broad; shell thick and transparent; stained yellowish with fecal ma- 
terial when in feces, but colorless in uterus; protoplasm unsegmented and 



THE FECES 



149 



.coarsely granular; covered with a mammilated albuminous envelope which 
may be lost.^ 

(2) Ascaris mystax. 

Synonyms. — Ascaris canis; ascaris lumhricus canis; ascaris teres; ascaris 
caniculce; ascaris cati; ascaris tricuspidata; ascaris felis; ascaris werneri; 
ascaris marginata; ascaris alata; and fusaria mystax. 

The male parasite is whitish or slightly brownish; 40 to 60 mm. long, i 
mm. thick; anterior end usually curved, with lateral cuticular expansions 
making the end look somewhat arrow-like; mouth terminal with three nearly 
equal lips with denticulate margins; at base of superior lip two papillae, on 
inferior hp one ordinary and two minute papillae; posterior end curled and 
with lateral cuticular alar expansions, and on each side of cloacal aperture 26 
papillae, of which live are postanal. 




Fig. 41. — Ascaris mystax. 
A, Male; B, female; C, anterior extremity, enlarged and shown from dorsum to exhibit 
the lateral wing-like cuticular expansions; D, same sho\ving in profile. (Tyson after 
Railliet.) 



The female worm is 120 to 180 mm. long; anterior end and general appear- 
ance as in male; posterior end straight, terminating obtusely; vulva at an- 
terior fourth of body length; anus subterminal. 

The ova are almost spherical; 68 to 72 microns in diameter; shell thin 
with thin albuminous envelope showing an alveolated surface. ^ 

(3) Oxyuris vermicularis. 

Synonyms. — Ascaris vermicularis; fusaria vermicularis; ascaris grcecorum; 
pin-worm; threadworm; seat-worm. 

The male is whitish in color; 3 to 5 mm. long, 0.3 to 0.4 mm. thick; 
cuticle transversely striated and at head end showing a vesicular swelling 
along the dorsal and ventral median lines; lateral lines distinct; mouth 

t^ See Foster, Jour. ParasitoL, 1914, I, 31; Perret and Simon, Jour. A. M. A., 1917, 
LXVIII, 244; Ransom, Ibid., 1919, LXXIII, 1210. 
2 See Beisele, Miinch. med. Wchnschr., 191 1, LVIII, 2391; Flury, Arch. f. exper. Path, 
u. Pharmakol., 1913, LXVII, 294. 



I50 



DIAGNOSTIC METHODS 



a 



terminal, witR three retractile lips; esophagus with distinct bulb; posterior 
end conical, curved ventrally, with six pairs of papillae and slight cuticular 
expansion on each side; one spicule hooked at free end. 

The female is lo mm. long, 0.6 mm. thick; 
anterior end and general appearance as in male; 
posterior end straight, extended to a long mucro- 
nate tail; vulva at anterior third of body length. 
The ova are oval, flattened on one side with a 
characteristic asymmetry, 50 microns long and 16 to 
20 broad; shell thin; colorless. 

This parasite inhabits the rectum and colon, 
but it may travel even into the stomach. The ova 
are rarely found in the feces, except in the mucus or 
about the anus.^ 

(b) Angiostomidae. 
Strongyloides intestinalis. 

Synonyms. — Anguillula intestinalis et stercoralis; 
leptodera intestinalis et stercoralis; pseudorhabditis 
stercoralis; rhabdonema strongyloides; rhabdonema 
intestinalis. 

This organism is found in two different forms : 
the first dioic and free, the second parasitic, as 
parthogenetic females. The parasitic form lives in 
the upper intestinal tract of man; is 2.5 mm. long, 
cylindrical, with pointed tail end, cuticle smooth; 
mouth simple with four lips; long, slender, cylin- 
drical esophagus reaching one-fourth of the length 
of the worm; anus close to tail; vulva at posterior 
third, containing yellowish-green oval ova, 50 to 58 
microns long and 30 to 34 microns broad. The 
larvae develop in the intestine and are passed in the 

Y-^Q -2 Oxvuris vermi- ^^^^ material. These larvae are at first from 200 

cularis: to left, female; to to 240 microns in length, but increase to two or 
enlargedr^' ^ ,^' anuf j^'^^o! ^hree times this length. The larv^ differ essentially 
mouth; v, vulva. (Ty^ow from the parent in having a rhabditiform esophagus. 
after Braun.) j^ ^-^^ discharged feces at about 3o°C. these de- 

velop with one moulting of the cuticle to a free-living generation with 
separate sexes. 

In this free sexual generation the worms are smooth, cylindrical, and 
tapering, with pointed tail-ends; the mouth is the same as in the parasitic 
form; esophagus rhabditiform with its anterior portion long and with the pos- 
terior pyriform and containing a Y-shaped chitinous armature; anus at base 
of tail; male with tail curved and two spicules, body length 0.7 mm.; female 

1 See Crowell and Hammack, Philippine Jour. Sc, 1913, VIII (B), 157; Willets, Ibid., 
1914, IX, 233; Fracker, Jour. Parasitol., 1914, I, 22; Aschofif, Berl. klin. Wchnschr., 1914, 
LI, 1504; Suzuki, Surg. Gyn. and Obs., 1915, XXI, 702. 




r 



THE FECES 



151 



I mm. long, with straight pointed tail, vulva a little back of the middle; ova 
few, yellowish, ellipsoid, thin-shelled, 70 by 45 microns in size, sometimes 
hatching in the uterus. The larvae of this generation look much as their 
free parents, are at first 0.22 mm. in length, but grow to 0.55 mm., then moult 
and assume a filariform or strongyloid character like that of the parasitic 
grandparent. These gain access to the intestine of a new host in an unknowrk 
manner or shortly die. 




Fig. 43. — Strongyloides intestinalis; on the left, a gravid female from human, intestine 
(natural size 2.5 mm.). In the middle, a rhabditiform larva from fresh fecal matter, X 120; 
to the right, a filariform larva from culture, X120. (Tyson after Braun.) 



These worms may be found throughout the upper gastrointestinal tract,, 
especially in the duodenum and upper part of the jejunum. The time elaps- 
ing between infections with the filariform larvae of the sexual generation and 
the appearance of the rhabditiform embryos of the parasitic type in the stools 
is between two and three weeks. The parthogenetic female types are usually 
called the strongyloides intestinalis, while the free sexual form is styled 
strongyloides stercoralis. 

This form is found widely distributed in Indo-China, the East Indies,. 




152 DIAGNOSTIC METHODS 

Africa, Europe, and North and South America. The mode of transmission 
to the second host is probably through the means of unfiltered water or of 
unclean, uncooked vegetables J 

(c) Trichotrachelidae. 
(i) Trichiuris trichiura. 

Synonyms. — A scar is trichiura; trichocephalus trichiurus; trichocephalus 
hominis; trichocephalus dispar; trichocephalus mastigodes; whip-worm. 

The male worm is 35 to 45 mm. long; whitish; anterior three-fifths slender 
and thread-like; posterior two-fifths thicker, cylindrical, 
terminally rounded and curled; anus terminal; single spicule 
in a tubular sheath containing small spinules. 

The female parasite is 35 to 50 mm. long; shape as in 
male for front and body; posterior extremity straight, 
bluntly pointed terminally; vulva at beginning of thick pos- 
terior portion of body. 

Fig. 44.— Tri- The ova are very characteristic, brown, oval, thick- 

cmuris trichiura, n 1 • , ^ i ^ ■ - ^ ^^^ 1 

natural size: A, walled, with a colorless shimng button-like protuberance at 
Male; B, female, each pole. The eggs are 50 to 54 microns long and 23 
microns wide, with an unsegmented yolk. Occasionally 
these eggs show variations in shades of brown, some being very much lighter 
than others.'^ 



(2) Trichinella spiralis (trichina spiralis). 

The male worm is 1.4 to 1.6 mm. long and 0.04 mm. thick; cylindrical 
in shape; anterior end tapering, posterior end gradually and slightly thicken- 
ing and terminating in a bifid extremity with two lateral somewhat conical 
tail appendages; cloacal aperture between these, which form a sort of bursa; 
back of cloacal aperture two pairs of papillae. 

The female worm is 3 to 4 mm. long; anterior end as in the male; posterior 
end nearly of same thickness to tail, which is rounded; anus terminal; vulva 
at anterior fifth of body; viviparous. 

The larvae when born are 90 to 100 microns in length, obtuse anteriorly, 
posteriorly prolonged to a pointed tail; when encysted as "muscle trichinae" 
the larvae measure about i mm. long and 0.04 mm. in thickness, tapering 
anteriorly, more thick and obtuse posteriorly with complete organization as in 
the adult and showing the characters of the different sexes. 

This parasite or its larvae are rarely if ever found in the stools. In its 
adult sexual stage it infests the intestinal tract of man and a number of mam- 
malians, gives origin to a large number of larval worms, after which the adults 
die. The larvae pass through the muscular wall of the intestine and are car- 
ried by the blood-current into various muscles of the host. Here they pass an 

^ Barlow, Interstate Med. Jour., 1915, XXII, 1201; Wachenheim and Bernstein, Jour. 
A. M. A., 1916, LXVI, 1092. 

2 See De Buys and Dwyer, Am. Jour. Dis. Child., 1919, XVIII, 269. 



THE FECES 



153 



indefinite encysted stage, a capsule forming around them and becoming 
calcified.^ 




Fig. 45. — Trichinella spiralis: a, Gravid female, "intestinal trichiura;" E, embryos; 
G, vulva; Ov, ovary; b, adult male, "intestinal trichiura;" T, testicles; C, young larva; 
d, larva in musculature; e, encapsulated larva in muscle. {Tyson after Braun.) 

Herrick and Janeway,^ Packard,^ and Cross^ found the embryo in the 

^ See Flury, Arch. f. exper. Path. u. Pharmakol., 1913, LXIII, 164; Gruber, Munch, med. 
Wchnschr., 1914, LXI, 645; Van Cott and Lintz, Jour. Am. Med. Assn., 1914, LXII, 680; 
Herrick, Jour. A. M. A., 1915, LXV, 1870; Bloch, Ibid., 2140, reports the finding of the lar- 
vae in the Cerebrospinal fluid; Bloch, 111. Med. Jour., 1916, XXIX, 369; Lintz, Jour. A M. 
A., 1916, LXVI, 1856; Cummins and Carson, Ibid., 1856; Salzer, Ibid., 1916, LXVII, 579; 
Cummins and Carson, Ibid., 806; Stiles, Ibid., 191 7, LXVIII, 685. 

2 Arch, Int. Med., 1909, III, 263. 

^ Jour. Am. Med. Assn., 1910, LIV, 1297. 

* Arch. Int. Med., 1910, VI, 301. 



154 DIAGNOSTIC METHODS 

blood, the latter in blood from an ear puncture. The blood was laked with 3 
per cent, acetic acid, was centrifuged, and the sediment examined as outlined 
by Staubli.i The chief means of infection of man with the trichinella 
spiralis is through the eating of insufficiently cooked pork, especially ham. 

(d). Strongylidae. 

(i). Uncinaria duodenalis. 

Synonyms. — Ancylostoma duodenale; strongylus quadridentatus; dochimus 
ancylostomum; sclerostoma duodenale; strongylus duodenalis; dochmius duo- 
denalis, and European hook-worm. 

The male parasite is whitish or blotched posteriorly with brownish when 
the intestine contains blood; 8 to 10 mm. long; cuticle finely striated trans- 





FiG. 46. — Tail, with expanded bursa, of Fig. 47. — Anterior 

male uncinaria duodenalis. {Tyson.) end, showing mouth 

parts, of uncinaria 
duodenalis, dorsal 
view. {Tyson.) 

versely; tapering to a blunt point anteriorly and with head curved upon the 
dorsum so as to give a slightly hooked anterior end; on each side of the median 
line on the ventral side of oral border two hook-like chitinous teeth and on 
dorsal border on each side of the median line one less curved chitinous tooth; 
with a dorsal conical tooth extending along back of oral cavity from the base 
of the cavity; in the oral cavity about the esophageal opening a delicate arma- 
ture consisting of two dorsal and two ventral lancet-like pieces; posteriorly the 
body ends in an abruptly pointed tail in a copulatory bursal expansion of the 
cuticle, this having one dorsal and two lateral lobes ; in the folds of the bursa 
one dorsal subdivided muscular ray, each division ending tridigitally, and on 
each side symmetrically placed an undivided dorsolateral, a divided lateral, 
undivided lateroventral, subdivided ventral and undivided small subventral 
muscular rays; cloacal aperture superterminal; two equal spicules. 

The female worm has the general appearance of the male and is shaped 
anteriorly like it; 12 to 18 mm. long; posteriorly tapering to a finely pointed 
tail; anus sub terminal; vulva about the posterior third of the body length; 
two uterine and ovarian tubes. 

1 Kolle and Wassermann's Handbuch der pathog. Mikroorg., 1913, VIII, 73. 



THE FECES I 55 

The ova are colorless, elliptical, thin-shelled with unsegmented or early 
segmenting material, '50 to 60 microns long and 30 microns broad. 

This worm has a wide distribution both in tropical and subtropical coun- 
tries. Its habitat is in the duodenum, jejunum, and upper part of the human 
ileum. Infection with this organism is known as uncinariasis or ancylostomia- 
sis.^ A particularly severe type of anemia is set up by this parasite both 
through the influence of loss of blood and the elaboration of toxic hemolytic 
material by the parasite. The eggs of this organism are frequently found in 
the stools and should be carefully searched for in every case of severe anemia. 
The mode of infection by these organisms may be direct ingestion of dirty 
water or unclean vegetables, but it is probable that the most frequent method 
of infection is through the skin, these larvse attaching themselves to the feet of 
people walking in infested sand or water. The larvae penetrate the skin and 
make their way through the blood- and lymph-currents to the lungs, whence 
they penetrate to the air-passages and are supposed to be carried upward to- 
ward the mouth by the bronchial mucus and are then swallowed. 

(2). Uncinaria americana^ (American Hook-worm). 

The male parasite differs from the former organisms discussed in being of 
smaller size (6 to 9 mm. long and more slender than the duodenalis), in the 
smaller size and more conical shape of the head, in having no hooklets on the 
oral rim, but instead on each side a large ventral and smaller dorsal chitinous 
lip, extending from the rim toward the median line; in a greater prominence 
and projection into the oral cavity of the dorsal conical tooth; in the smaller 
size of the copulatory bursa, its dorsal lobe being subdivided and the ventral 
margin being extended so as to form an indefinite ventral lobe; and showing 
the dorsal muscular ray of the bursa divided, each division ending in a bipar- 
tite tip. 

The female worm differs from the duodenalis in being shorter and more 
slender (8 to 15 mm. long) , with similar differences of the anterior end as 
above outlined for the male; vulva just in front of the middle of body length 
instead of at the posterior curve as in the uncinaria duodenalis. 

The ova of this parasite are somewhat larger (68 to 70 microns long and 
38 to 40 microns broad) than those of the uncinaria duodenalis, but are other- 
wise similar. 

Stiles has found that these parasites^ are the common cause of the frequent 
" anemia of the South." Smith believes that uncinariasis exists in every case 
in which "ground-itch" has occurred within eight years and that the disease 
is rarely ever present in those who have not had this condition during that 

^ See Bruns, Kolle and Wassermann's Handbuch, 1913, VIII, 41; Soltan, Lancet, 1919, 
II, 690. 

2 Also called Necator americanus. See Stitt, Jour. Am. Med. Assn., 191 2, LIX, 1706; 
also Stiles, Pub. Health Reports, 1913, XXVIII, 7; also, Ashford, War Dept., Office of 
Surgeon General, Bull. 2, 1913, 59 and 72; Ferrell, Jour. Am. Med. Assn., 1914, LXII, 1937; 
Billings and Hickey, Jour. A. M. A., 1916, LXVII, 1908; Frick. Am. Jour. Med. Sc, 1919, 
CLVII, 189. 

3 See Glover, Jour. Am. Med. Assn., 1912, LVIII, 1837, for a discussion of uncinariasis 
in oriental immigrants. 



156 



DIAGNOSTIC METHODS 



period. This would point to the general transmission of infection through 
the skin. The uncinaria duodenalis has long been known as the cause of the 
so-called Egyptian chlorosis, tunnel-workers' anemia, brick-layers' anemia, 
and other conditions necessitating work in low-lying watery places. 

Pseudoparasites. 

It not infrequently happens that extraneous substances are found in the 
feces which very closely simulate a parasite or its ova in appearance. These 
substances are, for the most part, food residues and should be carefully differ- 
entiated by applying tests for cellulose, which will show in all vegetable cells. 

Stiebel has described, under the name of diacanthos polycephalus, a 
fragment of the woody portion of a bunch of raisins. Sultzer describes a mul- 
berry seed as a vesicular worm under the name of ditrachyceros rudis. Basti- 





^^^.-^-: 



Fig. 48. — Tail, with expanded 
bursa, of male uncinaria ameri- 
cana. (Tyson.) 



Fig. 49. — Anterior end 
of uncinaria americana, 
showing mouth parts 
(dorsal view). (Tyson.) 



ani considers the larynx of a bird which he found in the fecal material as being 
a biped worm, under the name of sagitula hominis. Scopoli regards a frag- 
ment of the trachea of a bird as an entozoon form under the name of physis 
intestinalis. A pupil of Moquin-Tandon describes a strip of lettuce under 
the name of striatula, regarding it as a worm intermediate between the ascaris 
and the oxyuris (Guiart and Grimbert).^ 

Perhaps the most frequent pseudoparasite of the feces is the pulp of an 
orange, it shows in the feces in the form of large oblong masses terminated 
by two slender extremities, one of which ends in a sort of parenchyma. These 
vesicular masses are the large cells which secrete the orange juice and which 
are generally found intact in the fecal material. These have been frequently 
mistaken for hydatids or for parasitic ova. Certain spores, such as those 
of the truffle or of lycopodium, have not infrequently been mistaken for para- 
sitic eggs. Moreover, one may find in the fecal material the pollen of the 
coniferous plants which very closely simulates parasitic ova. The spines 
which form the down on certain fruits, such as raspberries, strawberries, 
peaches, and quinces, so closely resemble parasites that careful study is in 
some cases essential. 



^ Francaviglia (Policl'nico, 1915, XXII, 1052), reports the presence of blood clots simu- 
lating parasites. 



THE FECES 



157 




lO.M 20. ;iO. ^O. SO. 6O. 70.. 



I 



so. 100. 110. 120. I30. I40. ISO. 160. 170. 180. ISO. ZOOi 



Fig, 50. — Parasitic bodies, ova, and larvae met in human feces; color approximate^only. 
(Tyson.) 



1. Larval strongyloides intestinalis. 9. Ovum 

2. Ovum of fasciola hepatica. 10. Ovum 

3. Ovum of taenia nana. 11. Ovum 

4. Ovum of uncinaria duodenalis. 12. Ovum 

5. Ovum of uncinaria americana. 13. Ovum 

6. Ovum of taenia saginata. 14. Ovum 

7. Ovum of taenia solium. 15. Ovum 

8. Ovum of opisthorchis sinensis. 16. Ovum 



of opisthorchis felineus, 

of cotylogonimus heterophyes. 

of taenia cucumerina. 

of ascaris lumbricoides. 

of dicrocoelium lanceatum. 

of bothriocephalus latus. 

of trichiuris trichiura. 

of oxyuris vermicularis. 



158 DIAGNOSTIC METHODS 

It is, therefore, wise in all cases before pronouncing a finding as one of 
a parasite to be perfectly sure of your ground. 

BIBLIOGRAPHY 

1. Braun. Die tierischen Parasiten des Menschen. Wiirzburg, 1908. 

2. Briancon. De L'Ankylostomiase. Lyon, 1904. 

3. Cammidge. Feces of Children and Adults. New York, 1914. 

4. Craig. The Parasitic Amebas of Man. Philadelphia, 191 1. 

5. Hecht. Die Faeces des Sauglings und des Kindes. Wien, 19 13. 

6. Hemmeter. Diseases of the Intestines. Philadelphia, 1902. 

7. Lynch. Coprologia. Buenos Ayres, 1896. 

8. MussRAVE AND Clegg. Amebas: Their Cultivation and Etiologic Significance. 
Manila, 1904. 

9. NoTHNAGEL. Beitrage zur Physiologie und Pathologic des Darmes. Wien, 
1895. Krankheiten des Darmes. Wien, 1900. 

10. VoN Oeeele. Technik der chemische Unterschung des menschlichen Kothes. 
Leipzig, 1908. 

11. Schmidt und Strasburger, Die Faeces des Menschen. Berlin, 1915. 

12. Van Ledden Hulsebosch. Makro- und mikroskopische Diagnostik der mensch- 
chen Exkremente. Berlin, 1899. • 



PLATE VIII. 









f 



aJ^ 



i( 



Katharine HiU 



Vegetable Cells found in Feces. (After Schmidt and Strasburger.) 



CHAPTER V 
PARASITES 

I. General Considerations 

In the previous portions of the work, the writer has introduced those 
parasites which are more particularly related to the parts under discussion. 
There are, however, a large number of organisms which do not fall naturally 
within the scope of any of the chapters outlined for this book. These will, 
therefore, be discussed in general without much regard to distinct classifica- 
tions, as many of the subdivisions have been treated previously. This discus- 
sion is taken, for the most part, from Tyson. ^ 

II. Trematodes (Fluke-worms) 

Flukes are naked and unsegmented flat worms, usually of the shape of a 
leaf or of the tongue (occasionally pyramidal or elongated and more or less 
cylindrical), provided with incomplete digestive canal (without anus), pos- 
sessing one or more suckers and occasionally hooklets; with but few exceptions 
hermaphroditic and, as a rule, presenting a complicated series of metamor- 
phoses in their development.^ 

In structure it is customary to speak of the surface upon which the genital 
pore opens as the ventral; this surface commonly shows also the orifices of 
the mouth and one or more suckers. On the dorsal surface in many occurs 
the opening of a small canal, spoken of as Laurer's canal, of unknown func- 
tion. The surface of the body is covered by a fairly thick and firm cuticle, 
often provided over variable areas with small spines or tubercles. Beneath 
the cuticle over its internal surface is spread the superficial muscular layer 
(not showing the structure of muscle of higher animals, however), with longi- 
tudinal, circular, and diagonal fibers, while within this along the borders is 
met the parenchymatous muscle. The general internal tissue of the body, 
spoken of as the parenchyma, is a fine reticular connective tissue which 
closely surrounds the various organs. 

The suckers of trematodes vary in number and arrangement on the ante- 
rior and posterior extremities, the ventral surface and its borders, and in a few 
cases also on the dorsal surface. Usually the oral opening is surrounded by 
such a sucker and in addition, in the forms Ukely to be met with in man, on the 
ventral surface some distance posterior to the oral sucker is a second, known 
as the ventral sucker or acetabulum, in the median line. Not infrequently in 
the lining of these suckers, on their lips, or on the cuticle close to the lips, 
chitinous hooklets are to be found. 

1 See Ward, Illinois Med. Jour., 1912, XXII, 417; Cort, III. Biological Monographs, 1915, 
I, No. 4. 

2 See Stunkard, Jour. ParasitoL, 1916, III, 21. 

159 



i6o 



DIAGNOSTIC METHODS 



The alimentary system consists of a mouth, opening in the oral sucker and 
situated terminally or on the ventral surface of the anterior end of the worm. 
This cavity continues into a dilated tube with thick walls, the pharynx, this 
extending posteriorly by a short, straight, and usually narrow esophagus, 
which divides in the anterior portion of the body into the two intestinal tubes 
or ceca. At the posterior extremity of the body is a small orifice, the excre- 
tory pore, which serves as outlet for a series 
of more or less complex canals for the con- 
vection of the fluid waste, the arrangement 
representing a low nephridial apparatus, 
while the mouth serves as an anus. The 
reproductive system is highly developed, 
showing numerous minor variations in the 
different genera and species. 

Adult flukes are parasitic upon a wide 
range of animal life, including the higher 
animals, fish, amphibia, reptiles, and birds, 
living as ectoparasites and endoparasites. 
The varieties affecting man are compara- 
tively few. The most common parts of the 
human body to be infested are the intes- 
tines, gall-ducts, respiratory tubes, and 
blood-vessels. 



{a) Fasciolidse. 
(i) Fasciola hepatica. 

Synonyms. — Distomum hepaticum; dis- 
tomum cavim; fasciola humana, cladoccelium 
hepaticum; common liver-fluke. 

A comparatively large fluke, measuring 
20 to 50 mm. long and 8 to 13 mm. wide, of 
Fig. 51. — Showing the sexual glands leaf shape, with anterior extremity pro- 
suckSrA^nSnaTLcif I)*^, vitemne lodged into a small cone; greatest width of 
glands; Dr, ovary; Ov, uterine canal; body about the anterior third of length; 
vlxTv^^iifdnafcp^^^ liglit brown color; cuticle provided with 

5", ventral sucker. {Tyson after Br aun.) alternating transverse rows of spines, ex- 
tending on ventral surface to the posterior 
level of testes, but not as far posteriorly on the dorsal surface; the oral 
sucker at the anterior end of cephalic cone, inclining to ventral surface, 
I mm. in diameter; ventral sucker near anterior end behind cephalic cone, 
1.6 mm. in diameter; well developed larynx and short esophagus; intestinal 
branches extending nearly to the posterior extremity of the worm, approach- 
ing the median line posteriorly with few median and numerous lateral 
branches; excretory pore at posterior extremity, with well-developed system 
of excretory tubes; genital pore in the median line anterior to the ventral 
sucker; two large highly branched testes, mostly posterior to the transverse 




PARASITES 



l6l 



vitelline duct; ovary single, branched, lying in front of testes and to one side 
of the median line; the uterus coiled into a rosette, showing as a brown spot 
just back of the ventral sucker on the ventral surface; viteUine glands numer- 
ous, ranging along each lateral border from 
the level of the ventral sucker to the pos- 
terior extremity of the worm; vitelline 
ducts running transversely at about the 
end of the anterior third of the body. The 
ova are yellowish-brown, oval, operculated, 
and measure 130 to 145 microns in length 
and 70 to 90 microns in width. 

This fluke is a common one in most 
mammalians and has a wide geographical 
distribution over the world, being found not 
infrequently in America. Its usual habitat 
is the gall-ducts, but it has been seen in the 
gall-bladder, intestines, in the portal and 
other venous channels and in subcutaneous 
cysts. The ova appear in the feces and are 
the chief means of diagnosis of this condi- 
tion. The embryo which develops from 
the ovum completes its developmental cycle 
in the body of a snail, the Limnaea trunca- 
tula. From the body of the snail the cer- 
cariae escape in the water and become at- 
tached to grass or other aquatic material, 
which is taken in by the animal. Infec- 
tion in man is quite rare, about 32 cases 
having been reported in the literature. 



h- 




(2) Fasciolopsis buski. 



crassum. 



Fig. 52. — Fasciolopsis buski; a, oral 

sucker; &, acetabulum; c, cirrus pouch; 

Synonyms. — Distomum buski; distomum d, vitelline glands; e, "shell gland;" 

/"and g, posterior and anterior testicles; 
. . . T , ^> ovary; i, cecum; k, uterus. {Tyson 

The length of this organism is variable, after Braun.) 

ranging between 24 and 70 mm.; breadth 

5.5 to 14 mm.; lance-shaped; narrowing more rapidly anteriorly than poste- 
riorly, maximal width about the middle of the length; no cephalic cones; 
brownish in color; cuticle without spines; oral sucker small and placed on 
ventral surface of anterior extremity; ventral sucker two or three times as 
large as the oral, placed near anterior end and showing a saccular distention 
extending posteriorly; very short esophagus, back of the fairly developed 
pharynx; the two ceca without branches; genital pore at the anterior quarter 
of the acetabulum; cirrus pouch large; testicles branched; in posterior part of 
body one back of the other; uterus in anterior half of body, tortuously coiled; 
ovary at middle of length of body, to the right of median line; Laurer's canal 
present; vitelline follicles numerous along lateral margin from the level of the 



l62 



DIAGNOSTIC METHODS 



ventral sucker to the posterior extremity; the transverse vitelline ducts at 

the equator of body. The ova are brownish, ovoid, operculated, and measure 

125 microns in length and 75 in width. 

Very Httle is known of the intermediate stage and of the host of Busk's 
intestinal fluke. This has been found in the small 
intestine of man and probably arises from eating 
infected food. 

(3) Opisthorchis felineus. 

Synonyms. — Distomum conus; distomum lanceo- 
latum (Siebold); distomum sihiricum; distomum tenui- 
colle; the European cat- fluke. 

Variable in size according to the state of contrac- 
tion, 8 to II mm. long and 1.5 to 2 mm. broad; yellow- 
ish-red and nearly transparent; flat and lanceolate; 
anterior end constricted and attenuated into a cone; 
posterior end more obtuse; cuticle without spines; oral 
sucker toward the ventral surface at anterior extremity ; 
ventral sucker at base of cone about one-fourth of 
body length; pharynx and esophagus of equal length; 
ceca comparatively straight and unbranched, reaching 
nearly to the posterior extremity and often seen filled 
with blood; excretory pore terminal; its tubular vesicle 
winding in the median line between the testes and 
branching in front of the anterior testes; testes in the 
posterior part of body, the anterior four-lobed, the 
other five-lobed; cirrus and pouch absent; genital pore 

chis" feTineus- from livqr ^^ ^^^ median line in front of the ventral sucker; 

of cat; 10 X I. {Tyson slightly lobate ovary in the median line anterior to the 
after Braun.) ^ , , , . . . , - 

testes; receptaculum seminis promment; uterus an- 
terior to the ovary and testes coiled in the middle third 
of the body; vitelline follicles occupy about the middle third of the body, be- 
ginning anteriorly at the level of the ventral sucker. The ova are oval in 
shape, operculated, and measure 30 microns in length and 11 in width. 

This worm has been found in the gall-ducts of man and other animals, 
especially in Russia, Siberia, Hungary, and Japan. 
(4) Opisthorchis sinensis. 

Synonyms. — Distomum sinense; distomum spathulatum; distomum hepatis 
endemicum seu perniciosum; distomum hepatis innocuum; distomum j a ponicum; 
the Japanese or Chinese liver-fluke. 

This parasite measures 10 to 20 mm. in length and 2 to 5 mm. in breadth; 
long and lanceolate; reddish and nearly transparent when fresh; cuticle with- 
out spines; oral larger than ventral sucker, on the ventral face of anterior 
extremity; ventral sucker about one-fourth of body length posterior to former; 
pharynx and esophagus small and a bifurcation of the latter close to oral 
sucker; ceca unbranched, reaching close to the posterior extremity: excretory 
system as in the previous species; in the posterior fourth of body the two 




PARASITES 



163 



testes, one in front of the other, with from four to six dendritic branches; 
no cirrus or pouch; genital pore in the median line just in front of the aceta- 
bulum; ovary tri-lobed and placed just anterior to a large gourd-shaped 
receptaculum seminis both in the median line and anterior to testes; uterus 
well- developed and coiled in the middle area of the body between the ovary 
and ventral sucker; vitelline follicles in the marginal fields along the middle 
third of the body. The ova are brown, oval, operculated, and measure 27 
to 30 microns in length and 15 to 17 in width. 

This fluke is comparatively common in Japan 
and eastern Asia and has been reported in 
America. It infests the gall-ducts and has been 
found both in the pancreatic duct and intestine. 
Little is known of the intermediate hosts. ^ 

Many other types of these distomata are met 
with in the liver of various animals and are occa- 
sionally found in man. Among these we find the 
distomum lanceolatum, distomum heterophyes, 
distomum conjunctum, and amphistomum 
hominis (gastrodiscus hominis). 

III. Nematodes (Round Worms). 

Eustrongylus gigas. 

Synonyms. — Ascaris canis et martis; ascaris 
visceralis et renalis; strongylus gigas; strongylus 
renalis; eustrongylus visceralis. 

The male worm is red in color; 14 to 40 cm. 
in length and 4 to 6 mm. in thickness; slightly 
tapering anteriorly ; mouth terminal ; with a hex- 
agonal orifice surrounded by six lips bearing 
papillae; cuticle thin and transparent, finely 
striated transversely; about 150 papillae along 
the longitudinal lines laterally; caudal extremity 
with an oval plate-like expansion serving as a 
bursa, its margin bearing small papillae and 
slightly indented dorsally and ventrally; single 
sexual spicule. 

The female parasite shows the general ap- 
pearance and head end as in the male; 20 to 100 
cm. in length and 5 to 12 mm. in thickness; caudal extremity obtuse, straight, 
with anus sub terminal; vulva 50 to 70 mm. posterior to mouth; single 
ovariari and uterine tube plicated from near the anterior end along the in- 
testine nearly to the anus, then returning to the vulva near the anterior 
end. 

The ova are brown, ellipsoid, with thick shell marked by external cribri- 
form depressions, 64 to 68 microns in length and 40 to 44 microns in breadth. 

1 See Gunn, Jour. A. M. A., 1916, LXVII, 1835. 




&—/ 



Fig. 54. — Opisthorchis sin- 
ensis; ventral surface, stretched; 
a, oral sucker; b, ceca; c, geni- 
tal pore; d, acetabulum; 
e, uterus; /, vitelline glands; 
g, ovary; h, receptaculum sem- 
inis; I, Laurer's canal; i, test- 
icles; k, excretory canal; m, 
excretory pore. (Tyson after 
Braun.) 



164 



DIAGNOSTIC METHODS 



This worm, which is more common in the dog, has been reported a 
number of times in man. It is the largest of the nematode worms and 
has its habitat in the pelvis of the kidney. Little is known of its life history. 

IV. Parasites of the Skin 

Arthropoda. 

These are bilaterally symmetrical segmented animals whose segments 
do not correspond, but vary in structure, and which primitively bear upon 
each segment a pair of jointed appendages. The segments are often more 
or less fused, thus forming special body-regions which may themselves be 
more or less fused together as well. The covering of these animals is a com- 




FiG. 55. — Eustrongylus gigas: female, natural size, in kidney of dog. 
{Tyson after Railliet.) 

paratively thick and strong cuticle which remains pliable between the seg- 
ments of the body and of the jointed appendages, but which commonly be- 
comes hard and shell-like from chitinous or calcareous material directly over 
the different body-segments and internodes of the jointed appendages. 
This arrangement requires that in the growth of the individual the firm ex- 
ternal covering should from time to time be shed, such changes taking place 
periodically and being known as moults. While each segment in the primi- 
tive animal is provided with a pair of jointed appendages, these in the indi- 
vidual species are often lost from this or that part of the body, or may remain 
rudimentary and inconspicuous or may take on special features of structure 
from the assumption of special function which causes their wide departure 
from the original and common type used for locomotion. The arthropods 



PARASITES 165 

commonly reproduce by ovulation, the development of the embryo to the 
adult often showing more or less complicated metamorphoses. The true 
parasite forms of the arthropoda thus far met in man are limited to the 
Arachnoids and Insects. 

{A) Arachnoidea. 
(a) Sarcoptes or acarus scabiei (the Itch Parasite). 

This parasite is oval in shape, is provided with horns and bristles, is barely 
visible to the naked eye, the male being from 0.2 to 0.3 mm. in length by 0.145 
to 0.19 mm. in breadth; the female is somewhat larger, showing a length 
of 0.33 to 0.45 mm. and a breadth of 0.25 to 0.35 mm. 

The female lies at the end of a burrow in the epidermis, in situations 





A B 

Fig. 56. — Acarus scabiei: A, female, dorsal view; B, portion of human epidermis, show- 
ing burrows with contained ova and young acarians. {Gould.) 

where the skin is' most delicate, as between the fingers, at the elbows, under 
the knees, and in the groin. In this burrow, which varies from a few milli- 
meters to a centimeter in length, the female deposits her eggs, after which 
she dies. The eggs hatch in from 4 to 8 days, and in about 14 days the 
larvae are sufficiently matured to make their own burrows. 

The disease is communicated either by the clothing or by personal con- 
tact. To demonstrate the parasite, the burrow is opened with a needle and 
the female pressed out on a slide, which is then covered and examined. 

{b) Demodex folliculorum. 

This parasite is very small, varying in length from 0.3 to 0.4 mm. It 
is somewhat cylindrical, tapering to an obtuse point at the posterior end. 
This parasite has its habitat in the sebaceous follicles, especially of the face 
and nose. 

(c) Leptus autumnalis (Harvest-bug). 

This parasite is also known as trombidium irritans or red jigger. This 
is a minute red parasite, from 0.3 to 0.5 mm. long, which has three pairs of 
legs, with rows of bristles upon its back and belly. It prevails in summer oni 



i66 



DIAGNOSTIC METHODS 



grass and plants and attaches itself to the skin of man by its hooklets. The 
condition of the skin due to the infection with this parasite is known as 
trombidiosis.^ 



(B) 
(a) 

(i 



Insecta. 
Hemiptera. 
) Pediculus capitis (Head-louse). 

The male is from i to 1.5 mm. long, the female 1.8 
to 2 mm. long. The color of the parasite varies some- 
what with the race of its host. In the Caucasian it is 
gray with a dark border, in the Negro and Chinamen it is 
much darker in color. The 
eggs are 0.6 mm. in length 
and are attached to the hairs, 
forming the so-called ''nits." 
These nits are whitish oval 
masses which are easily visible. 

This parasite, while usually 
found upon the hair of the head, 
may be found in other portions 
of the body. 2 The symptoms 
may be severe or very slight. 





Fig. 57. 
Demodex foUi- 
culorum: from 
dog, enlarged. 
{Tyson after 
Braun.) 



vestimenti 



Fig. 58. — Leptus autum- 
nalis; enlarged. {Tyson after 
Braun.) 



(2) Pediculus 
(Body-louse). 

This parasite is consider- 
ably larger than the former, being from 2 to 5 mm. long 
and whitish-gray in color, the back part of the body being 
wider than the thorax. The antenna are longer than are those of the head- 
louse. The eggs are from 0.7 to 0.9 mm. in length, about 70 being laid by 
each female. 

This parasite is found upon the clothing in which it deposits its eggs, 
especially about the neck, back and abdomen. 



(3) Pediculus pubis (phthirius inguinalis or Crab-louse). 

This parasite is smaller than the head-louse, grayish-yellow or grayish- 
white in color, the male being from 0.8 to i mm. in length, the female about 
1. 1 2 mm. in length. The eggs are pear-shaped, from 0.8 to 0.9 mm. in length 
and from 0.4 to 0.5 mm. in breadth. 

This parasite infests the parts of the body covered by the shorter hairs, 
such as the pubis, axilla, eye-brows, and chest. 

^ See Olson, Jour. Am. Med. Assn., 1915, LXIV, 2060; Nagayo, Miyagawa, Mitamura 
and Imamura, Jour. Exper. Med., 191 7, XXV, 273. 

2 Goldberger and Anderson (Pub. Health Rep., 191 2, XXVII, 297) show that typhus 
fever may be transmitted by this parasite as, also, by the body louse. See, also, NicoUe, 
Comte and Conseil, C. R. Acad. Sc, 1909, CXLIX, 486; Ricketts and Wilder, Jour. Am. 
Med. Assn., 1910, LIV, 1304; Goldberger and Anderson, Pub. Health Rep., 1910, XXV, 177; 
Ibid., 191 2, XXVII, 835; and Jour. Am. Med. Assn., 191 2, LIX, 514. 



PARASITES 



167 



(4) Cimex lectularius (acanthia lectularia or Bed-bug). 

While, strictly speaking, the bed-bug is not a parasite of man, yet as its 
habitat is the bed, bedding, and walls of the sleeping apartment of man, it 
may be considered as indirectly parasitic. It usually emerges at night from 




Fig. 59. — Pediculus capitis, X15. 

{Tyson after Br aim.) 




Fig. 60. — Pediculus vesti- 
menti, Xio. {Tyson after 
Braun.) 



its lodging for the purpose of securing its nourishment in the blood of its 
victims. 

This parasite is reddish-brown in color, oval in shape, from 4 to 5 mm. 
in length and 3 mm. in breadth. These insects, if crushed between slides 




Fig. 61. — Pediculus pubis. {Tyson after Braun.) 



or as more usual between the hand and a part of the victim's body, have a 
characteristic odor very much resembling kerosene. The blood is drawn from 
the victim by means of a long proboscis. The eggs are approximately 1.12 
mm. in length and require about 11 months for their development to the 
sexually ripe insect. These eggs are retained in the crevices of the bed, floors, 



1 68 



DIAGNOSTIC METHODS 



furniture, wall-paper, and other parts of the dwelling so that the complete 
removal of these eggs and parasites is a matter of some difficulty.^ 

That these insects have more or less importance from the standpoint 
of transmission of disease from one person to another must be remembered. 
Individuals vary, in their susceptibility to the bite of the bed-bug, some being 
indifferent to it while others are markedly affected by it. 




Fig. 62. — Pulex irritans, X14. {Tyson after Braun.) 



{b) Diptera. 
(i) Pulex irritans (Common Flea). 

The male is from 2 to 2.5 mm. in length, the female as much as 4 mm. 
It is a red or brownish-red insect, having a laterally compressed body, an oral 
haustellum, serrated soft mandibles, a tongue sheathed in an inferior labium, 
and a pair of labial four-jointed palpi. Each of the triple segments of the 
thorax bears a pair of five-jointed double-clawed legs. The female deposits 
her eggs, not on the human being, fortunately, but in the fissures, crevices, 
or holes of garments or furniture which may be accessible. 

(2) Pulex penetrans (Sand-flea or Jigger). 

This parasite is a minute, brownish-red, egg-shaped insect which pene- 
trates the skin of man. The female is the infecting insect and produces pain- 
ful irritation and even suppuration. 

Vegetable Parasites. 

(i) Achorion Schonleinii. 

This organism is the cause of the disease known as favus or tinea favosa. 
This fungus invades the root sheaths, the bulbs, and the shafts of the hair 
filaments of the scalp, but it also occurs upon the "non-hairy" portions of 
the skin and upon the nails. ^ The spores gain access to the deeper layers of 
the skin and develop around the hair-shaft, forming a characteristic yeUowish 
cup-shaped crust which has a peculiar mouse-like odor. 

1 See Rucker, Pub. Health Reports, 191 2, XXVII, 1854; Thomson, Ann. Trop. Med. and 
Parasitol., 1914, VIII, 19. 

2 See Foster, Jour. Am. Med. Assn., 1914, LXIII, 640. 



PARASITES 



169 



In searching for this parasite, a favus crust is softened by the addition 
of a few drops of water or dilute sodium hydrate solution and placed upon 
a slide and examined with the high-power dry lens. The hairs may also 
be examined in the same manner or may be stained by methods outlined in 
the discussion on Tinea tricophytina. 

The mycelial threads appear as narrow, flattened, ramifying, short or 
elongated, linear cells or tubes, which may be simple and empty, or be divided 
more or less regularly by transverse partition walls transforming the longer 
and simple into shorter and compound cells. The latter often contain in their 
cavities sporules clinging to either side, in which case the mycelial threads are 
termed sporophores. The conidia are encapsulated or are strung together 




Fig. 63. — Pulex penetrans: young female, enlarged. {Tyson after Braun.) 



like the beads of a necklace, and appear as round, oval, angular, or very 
irregularly contoured bodies. These mycelial threads branch at right angles, 
the spores measure from 3 to 10 microns in diameter (Hyde). 

(2) Trichophyton megalosporon endothrix. 

This organism is the cause of tinea circinata (herpes tonsurans, ring- 
worm of the body), and of tinea sycosis (hyphogenous sycosis, tinea barbae, 
ringworm of the beard, barber's itch). 

The trichophyton is composed of spores which vary greatly in size, but 
which, as a rule, are somewhat larger than those of the type next to be dis- 
cussed.^ They are frequently cuboidal, oval, or irregularly rounded, but 
their chief characteristic lies in their arrangement in lines or chains, extending 
up and down the hair shaft. The mycelium is found without, but never 
within the hairs (Hyde). 

These fungi may be stained by the method of Morris and Calhoun. The 
hair is first washed in ether to remove all fatty debris; it is then put for one 

1 Priestley (Ann. Trop. Med. and ParasitoL, 1914, VIII, 113) reports the presence'of the 
microsporon scorteum in a case of ringworm. See Ormsby and Mitchell, Jour. A. M. A., 
1916, LXVII, 711; Muijs, Nederl. Tijdschr. v. Geneesk., 1916, II, 1985. 



lyo 



DIAGNOSTIC METHODS 



or two minutes in Gram's iodin solution and is stained after drying for from 
one to five minutes in gentian- violet. It is again dried and treated for a min- 
ute or two with the iodin solution and for an equal length of time in aniline oil 
containing pure iodin, after which it is cleared with aniline oil, washed in 
xylol, and mounted in Canada balsam. 

(3) Micro sporon audouini (Trichophyton Microsporon). 

This parasite appears under the microscope chiefly in the form of a large 
number of round spores, irregularly grouped or massed about the follicular 
portions of the hair. Mycelial threads, large and branching, are often seen 




Fig. 64.— Achorion schonleinii, X 500 diameters. {Van Harlingen.) 

within the hair. The sheath of spores surrounding the hair is often con- 
tinued upward for J-fg to 3^^ inch above its exit from the follicle and may 
be recognized as a whitish or grayish coating of the hair. These mycelial 
threads are all within the hair proper, thus differing from those of the tricho- 
phyton which are never within the hair; after repeatedly dividing and sub- 
dividing they terminate on the outer surface of the shaft in fine filaments, at 
the extremities of which are the spores. This parasite is the cause of the dis- 
ease tinea tonsurans, or ringworm of the scalp. ^ 

(4) Microsporon furfur. 

This parasite is readily recognized by the microscopic examination of the 
scales scraped from the skin. Innumerable clustered spores, highly refractive 
and resembling in their circular and oval contours droplets of oil, are quite 
characteristic. The mycelial threads are not usually branched, but lie in a 
close network, among which sporophores are distinguishable, with conidia 
and terminal elements emerging at one extremity of the spore case. Both 

1 See Beeson, Jour, Cut. Dis., 1915, XXXIII, 731. 



PARASITES 



171 



if 



i0im 



Mm 






Fig. 6$. — Normal hair, X 900. 



1,1 ( 



(lll1!'3'|lr'«i:fl\(,: 



ll. 



'''4i 'e 









%\gwm 



m^ 



Fig. 66.— Hair showing trichophyton endo-ectothryx, X 900. 



172 



DIAGNOSTIC METHODS 



elements of this organism are more readily stained by the aniline dyes than 
are those of the trichophyton or favus. This organism is the cause of the con- 
dition known as tinea versicolor. 

(5) Microsporon minutissimum. 
This organism is the etiologic factor of erythrasma. It is characterized 
by the extreme delicacy and fineness of its threads and very minute spores. 
The threads are either simple, cylindrical bodies of variable size or they 
may exhibit partition septa, may divide dichotomously, and may terminate 
in hooked or knobbed expansions. The largest transverse diameter is 0.6 
micron; in length the mycelium presents the greatest variations. 




Fig. 67. — Hair showing microsporon audouini, X 900. 

(6) Blastomycetes. 
These organisms may be found in the cutaneous eruptions of the skin 
in blastomycosis and may be described as follows, according to Montgomery 
and Ormsby . In unstained preparation the organisms appear as round or oval 
bodies with a double-contoured highly refractive capsule. Within the cap- 
sule, in many instances, granules or spore-like bodies can be distinguished. 
The addition of a i to 10 per cent, solution of potassium hydrate to the 
specimen under examination facilitates the recognition of these bodies. In 
stained sections the double contoured, homogeneous capsule is usually sepa- 
rated from a finely or coarsely granular protoplasm by a clear space of vary- 
ing width. Vacuoles of different sizes are found in some organisms. In 
both pus and tissue, organisms in pairs or in various stages of budding are 
commonly seen.^ The parasite, as a rule, varies in size from 7 to 20 microns, 
though slightly smaller and much larger forms occur in some cases. 

1 See Whitman, Jour. Infect. Dis., 1913, XIII, 85. See a long series of articles by various 
authors in Arch. Int. Med., 1914, XITI, 509 &.; Wade, New Orleans Med. and Surg. Jour., 
1915, LXVIII, 287; Wade and Bel, Arch. Int. Med., 1916, XVIII, 103; Watts and Wells, 
Tr. Chicago Path. Soc, 1916, X, 92. 



PARASITES 



173 



The organisms are readily obtained in pure culture from unbroken ab- 
scesses, from miliary abscesses in the borders of the cutaneous lesions, and 
from the miliary nodules and abscesses in the deep-seated organs. The 
peculiarities of the cultures of blastomycetes must be looked for in other 
works. Microscopically, the organism obtained in culture appears at first 
as a fine, branching mycelium with a few small spore-like bodies. Later a 



► 







» 


^ 




# 








t 






% 


f 

/ 

„..» V 


\ 


_^y\ 




X.. 








. 


■■■■«<, 


t^.. 



Fig. 68. — Mycelial threads of blastomyces from old agar culture. 
(From photograph by W. A. Pusey.) 



large, segmented, often pod-like mycelium appears, together with large, round, 
or oval bodies with bud-like projections. 

The condition known as coccidioidal granuloma, which is especially ob- 
served in California, has often been confused in the literature with blasto- 
mycosis. It is, however, now well established that the etiologic factor of 
this disease is the coccidioides immitis, an organism somewhat similar to but 
differing distinctly in many respects from the blastomyces. This organism 



174 DIAGNOSTIC METHODS 

was first reported by Posadas and Wernicke, but was later classified by 
Rixford and Gilchrist and cultivated by Ophiils.^ 

(7) Sporothrix Schenckii. 

This organism is the cause of sporotrichosis and was first recognized 
by Schenck, Hektoen and Perkins.- Since its discovery a large number of 
cases have been identified in all parts of the world, especially in the middle- 
western portions of the United States. The lesions in this condition re- 
semble, at times, those of tuberculosis, while, occasionally, they appear 
more hke those of syphilis. The chief characteristics are the sharply de- 
fined, painless, cutaneous or subcutaneous abscesses which follow the lym- 
phatics and do not yield to the ordinary surgical procedures. In living tissues 
the sporothrix organisms appear as elongated or oval bodies fairly uniform in 




Fig. 69. — Budding forms of Sporothrix Schenckii in pus, X 1200. Gram's Stain. (Cour- 
tesy of Dr. D. J. Davis.) 

size and often showing distinct budding processes. The arrangement is usu- 
ally single, but frequently two or more are found end to end or arranged 
radially about a central spore. Branched mycelial filaments are not found 
in the living tissues but they develop abundantly on artificial media, form- 
ing, at their sides and extremities, numerous spores, oval in shape and differ- 
ing decidedly in appearance from the typical tissue forms. These latter 
forms may be found, however, in small numbers in artificial cultures (Davis). 
Smears from the lesions are made in the usual way. To obtain cultures 
proceed as follows: Some of the light yellow pus is taken, by aseptic m.ethods, 
from the suspected lesion, is transferred either to glucose or mialtose agar, 
or blood agar, and is kept at room temperature. After a few days small 
points appear from which circular growths with marked striations develop 
in 10 to 1 2 days. Later these may show a deep black pigmentation. Smears 

^ See MacNeal and Taylor, Jour. Med. Research, 1914, XXX, 261; Cooke, Arch, Int. 
Med., 1915, XV, 479; Brown and Cummings, Ibid., 608; Dickson, Arch. Int. jNIed., 1915, 
XVI, 1028; Cummins and Sanders, Jour. Med. Research, 1916, XXXV, 243; Lipsitz, 
Lawson and Fessenden, Jour. A. M. A., 1916, LXVI, 1365; Lipsitz, Jour. Missouri State 
Med. Assoc, 1916, XIII, 534; Pierson, Jour. A. M. A., 1917, LXIX, 2179, reports a case 
of torula infection in man; Helsley, Ibid., 1919, LXIII, 1697. 

2 See Gougerot, in Kolle and Wassermann's Handb. d. path. Mikroorg., 191 2, V, 211. 



► 



PARASITES 175 

made from these cultures show a typical sporothrix, the mycelium being 
coarse (2 /x broad) and forming a dense branching, septate network. Spores 
(4 to 5 M long and 2 to 3 /x broad) develop by budding either from lateral or 
terminal filaments or from the sides of the thread. The organism stains 
readily with the ordinary dyes and is, also. Gram-positive. ^ 
(8) Negri Bodies. 

Considerable discussion has centered about these bodies as the etiologic 
factor of rabies (hydrophobia), but now, thanks to the successful experiments 
of Noguchi^ in cultivating them, the subject must be regarded as practically 
settled in the affirmative. These bodies are found in almost all of the nerve 
cells of the central nervous system but are more numerous and more easily 
found in the cells of Ammon's horn, cortical tissue in region of the fissure of 
Rolando, and in the Purkinje cells of the cerebellum. They are also present 
in the salivary glands of the infected animal but are not readily detected 
therein. Their demonstration in the cells of the brain is regarded as equiva- 
lent to a positive diagnosis of rabies. As they have never been found in 
any condition other than hydrophobia, they must be regarded as specific. 

These bodies were originally described by Negri,^ although Galtier^ and 
Pasteur^ had previously demonstrated the infectious character of rabies. 
Since that time many workers have confirmed their findings and have 
added much to our knowledge of these bodies.^ 

They are found lying in the cytoplasm of the nerve cells or in their 
branches, occasionally outside, and appear as round, oval, triangular, pear- 
shaped, spindle- or sausage-shaped bodies. They vary in size from 0.5 m 
to 20 ;u in diameter, the size increasing with the stage of the disease. They 
show a hyaline cytoplasm with an entire margin and with one or more inner 
bodies (nuclei) having a more or less complicated and regular structure. 
There do not appear to be any vacuoles in the smear preparations but in the 
tissue specimens vacuoles are quite distinct (probably artefacts). Most of 
the bodies show chromatoid granules of varying size and number and ar- 
ranged about the nucleus like a ring. In stained specimens these granules 
are seen as basophilic granules, rods or circles. They are, therefore, properly 
classified as protozoan parasites. 

^ See Hamburger, Jour. Am. Med. Assn., 1912, LIX, 1590; Sutton, Ibid., 1913, LX, 115; 
Chipman, Jour. Cutan. Dis,, 1912, XXX, 339; Adams, Journal Lancet, 1912, XXXII, 395; 
Jour. Am. Med. Assn., 1913, LX, 1784; Taylor, Ibid., 1913, LX, 1142; Davis, Jour. Infect. 
Dis,, 1913, XII, 453; Hamburger, Illinois Med. Jour., 1914, XXV, 99; Dominguez, Med. 
Record, 1914, LXXXV, 608; Davis, Jour. Infect. Dis., 1914, XV, 483; Sutton, Jour. Am, 
Med. Assn., 1914, LXIII, 1153; Meyer and Aird, Jour. Infect. Dis., 1915, XVI, 399; Davis, 
Ibid., 1915, XVII, 174; Campana, Riforma Med., 1915, XXX, 925; Bolognesi, Policlinico, 
1916, XXIII, 129; Burovi, Russ. Vrach, 1916, XV, 876; Accame, Semana Med., 1916, 
XXIII. 680; Spoor. Jour. A. M. A., 1917, LXVIII, 1458; Moore and Davis, Jour. Infect. 
Dis., 1918, XXIII, 252. 

2 Jour. Exper. Med., 1913, XVIII, 314. See, also, Levaditi, C. R. Soc. de Biol._, 1913, 
LXXV, 505; Kraus and Barbara, Deutsch. med. Wchnschr., 1914, XL, 1507; Noguchi, Berl. 
klin. Wchnschr., 1914, XLI, 1931; Proescher, New York Med. Jour., 1914, C, 953. 

^Ztschr. f. Hyg., 1903, XLIII, 507; XLIV, 520; 1909, LXIII, 421. 

4 e. R. Acad. d. sc, 1879, LXXXIX, 444- 

^Ibid., i88i,XCII, 159. 

^ See Babes, Traite dela rage, Paris, 191 2; Proescher, Berl. klin. Wchnschr., i9i3,L, 633; 
Poor and Steinhardt, Jour. Infect. Dis., 1913, XII, 202; Luzzani, Ann. de ITnst. Pasteur, 
1913, XXVII, 907 and 1039; LeComte, Jour. Am. Med. Assn., 1914, LXIII, 1658. 



176 DIAGNOSTIC METHODS 

To demonstrate these Negri bodies in the brain cells of a suspected animal, 
the following method of Williams andLowden^ is recommended for diagnostic 
purposes. A fragment of the gray substance from the cerebral cortex in the 
region of the fissure of Rolando, another from Ammon's horn and a third from 
the cerebellum, is taken from a section made at right angles to the surface and 
placed on slides about i inch from their ends. A cover-glass is now pressed 
upon it until it is spread out in a moderately thin layer; the cover-glass is 
moved slowly and evenly over the slide, leaving the first portion of the slide 
clean. Use slight pressure in making the smear, pressing rather more on the 
edge of the cover-glass away from the end of the slide toward which the cover- 
glass is moving, thus driving more of the nerve tissue along the smear and 
producing more well-spread nerve cells. Dry in air and stain as follows. 

Fix the air-dried smears in methyl alcohol for five minutes. Pour on the 
slide the staining solution and allow to stand for one-half to three hours. The 
staining solution is made by adding 10 drops of Giemsa's stain to 10 c.c. of dis- 
tilled water made alkaline by the previous addition of i drop of i per cent, 
potassium carbonate solution. The longer period of staining is preferable, as 
overstaining does not occur even after 24 hours. Pour off the stain, wash in 
running tap- water for one to three minutes and dry with filter paper. If the 
smear be thick, dip it in 50 per cent, methyl alcohol before it is washed in water. 

The cytoplasm of the Negri bodies stains blue, while the central bodies 
and chromatid granules stain a blue-red or azur. The larger bodies have 
usually a somewhat darker blue tint than the smaller ones. The cytoplasm 
of the nerve cells stains blue, while the nuclei are red, the nucleoli are a dull 
blue and the red cells a pink-yellow. 

If it is desired to make a quick examination proceed as follows, although 
this method is not to be advised. Fix in methyl alcohol for five minutes and 
add equal parts of Giemsa's stain and distilled water for ten minutes. Wash, 
dry and examine. 

Mallory^ recommends the following technique. The smears made as 
above described are fixed in Zenker's solution for one-half hour; after being 
rinsed in tap-water they are placed successively in 95 per cent, alcohol, con- 
taining sufficient iodine to make a port-wine color, one-fourth hour, 95 per 
cent, alcohol one-half hour, absolute alcohol one-half hour, 10 per cent, 
aqueous solution of eosin for 20 minutes, rinse in tap-water, place in alkaline 
methylene-blue solution for 15 minutes, differentiate in 95 per cent, alcohol 
from one to five minutes, dry with filter paper and examine. The cytoplasm 
of the Negri bodies is a magenta, the central bodies and chromatoid granules 
are a very dark blue, the nerve cell cytoplasm a light blue, the nucleus a 
darker blue and the red cells a brilliant pink. 

Van Gieson^ recommends the following: To 10 c.c. of distilled water add 
3 drops of a saturated alcoholic solution of rose-aniline violet and 6 drops of 
Loeffier's methylene-blue solution. Fix the smears in methyl alcohol, pour 
the above stain on them and warm until steam rises. Pour off the stain, rinse 

' Jour. Infect. Dis., 1906, III, 452. 

2 Pathological Techmque, Philadelphia, 191 1. 

3 Centralbl. f. BakterioL, I. Abt. 1907, XLIII, 205. 



PARASITES 



177 



in water, and allow to dry. The stain must be prepared fresh each time as it 
is not permanent. The cytoplasm of the bodies is a deep and distinctive red, 
their inner structures are a dark blue, the nerve cell light blue and the blood 
cells a pale salmon red. ^ 

Noguchi^ has succeeded in cultivating these bodies from infected animals 
and has, in turn, reproduced rabies by inoculating cultures containing the 
granular, pleomorphic, or nucleated bodies. He has, then, recovered the 
granular and nucleated bodies from the brain of the inoculated animals. 
The specificity of these bodies would seem, therefore, to have been definitely 
established. See, however, Moon-^ and Williams.^ 



BIBLIOGRAPHY 

Carpenter. The Microscope and its Revelations. Philadelphia, 1901. 

Daniels. Laboratory Studies in Tropical Medicine. Philadelphia, 1903. 

Emery. Bacteriology and Hematology. Philadelphia, 1905. 

Hyde and Montgomery. Diseases of the Skin. Philadelphia, 1902. 

Jackson. Tropical Medicine. Philadelphia, 1907. 

Oertel. Medical Microscopy. Philadelphia, 1902. 

Pusey. Diseases of the Skin. New York, 1907. 

1 See Kotsevaloff, Russky Vrach., 1914, XIII, 616. 

2 Jour. Exper. Med., 1913, XVIII, 314. 
^ Jour. Infect. Dis., 1913, XIII, 232. 

^ Jour. Am. Med. Assn., 1913, LXI, 1509. 



r 



CHAPTER VI 
THE URINE 

I. General Considerations 

The examination of the urine is one of the most important features of 
dinical diagnosis. So constant are the physical and chemical properties of the 
normal urine that any marked abnormality is easily detected. The relation 
between the kidneys and the blood is so close that the kidneys soon excrete 
any abnormal substances which have found their way into the blood-current. 
For this reason we find in the urine the abnormal products of perverted metabo- 
lism of the system or of special organs. It is true that the urinary findings 
may be, in any special case, secondary to those of the blood or of clinical ex- 
amination, but in such conditions we may detect substances in the urine, 
which put us on our guard against making a specific diagnosis or point out 
the way to a correct differentiation. More or less marked changes in the 
character of the urine will occur whenever a pathologic condition exists any- 
where in the system. These changes may not always be sufficient to be of 
direct diagnostic value, but in many cases may settle a differential diagnosis.^ 

In the urine we find excreted the products arising from the metabolism 
of the various proximate principles, both of the tissues and the food. Know- 
ing the intake of such material, we are able from our examination of the urine 
to judge of the manner in which the system is handling the material brought 
to it. In recent years the study of the metabolism in various conditions has 
been so extended that determinations which a few years ago were unusual are 
now matters of almost daily routine. An examination of the urine will fre- 
quently reveal the presence of irregular digestive and absorptive powers of the 
intestines through the appearance of certain abnormal products of protein 
decomposition. Moreover, the study of the nitrogen partition of the urine 
is taking on increasing importance from day to day, so that the estimation of 
the factors determining this division should be possible by any one attempting 
to follow the metabolic activity of the system in any specified condition. 

When the oxidative powers of the system are lessened, we find the urine 
showing abnormal products as an indication of such deficiency. These 
products are more or less characteristic and may be determined with a great 
degree of exactitude. Thanks to the work upon metabolism in diabetes, for 
instance, we now know that the glycosuria is clinically not of as great im- 
portance as is the presence of many other abnormal products associated with 
the sugar; in other words, a glycosuria must not be considered as identical 
with diabetes. 

The system has a definite disintoxicating power toward certain noxious 

^ See Freeman, Jour. Am. Med. Assn., i9i4,LXIII, 1802; Diner, New York Med. Jour., 

1915, CI, 1007; KildufiFe, Arch. Diag., 1915, VIII, 383; Stark, Jour. Lab. and Clin. Med., 

1916, II, 134. 

178 



THE URINE 179 

substances, whether introduced from without or formed within. While the 
blood is of special importance in such processes as far as bacterial products are 
concerned, an examination of the urine will frequently reveal much informa- 
tion regarding the metabolic toxins or the medicinal poisons. The estima- 
tion of the conjugated glycuronic and sulphuric acids of the urine will throw 
much light on the degree of this activity. In this connection it may be 
mentioned that indican, a product of bacterial decomposition of protein in 
the intestinal canal, may be taken as a direct indicator of the degree of such 
decomposition, but that we must not assume that all of the conjugated acids 
have such an origin. 

Besides these general indirect points of interest, an examination of the 
urine will often reveal a direct anatomical lesion of the kidneys. Time was 
when we regarded the mere presence of albumin in the urine as indicative of a 
kidney lesion, but we know that a thorough clinical examination is necessary 
before a diagnosis is possible. Albumin may or may not mean kidney trouble 
and may even be purely physiologic. Too much stress can hardly be laid 
upon the necessity of closely associating the urinary findings in any condition 
with the clinical symptoms of the case. The writer will have much to say 
later regarding the various abnormalities of the urine, but he wishes to im- 
press at this point the fact that no finding, no matter how abnormal it may 
seem, should be considered absolutely pathognomonic, without taking into 
consideration the clinical manifestations of the case. An albuminuria, gly- 
cosuria, cylindruria, or pyuria may mean one thing at one time and another 
at a second period, so that the worker is cautioned against jumping at con- 
clusions. The urine will yield much information, providing the worker knows 
how to interpret his findings. A laboratory worker must be cautious in his 
attitude and report his findings without any attempt at interpretation, unless 
he is aware of the clinical history of the case in point. 

The writer must refer to works on physiology for the various theories 
which have arisen from time to time regarding the mechanism of secretion of 
the urine. Suffice it to say at this point that the urine is excreted through the 
activities of the kidneys, the water and salts being secreted by the glomeruli, 
while the majority of the excretory products are eliminated by the vital or 
selective activity of the epithelium of the renal tubules. It is evident, there- 
fore, that the physical and chemical characteristics of the urine will depend 
both upon the blood-pressure^ within the capillaries and the rate of flow 
through these vessels as well as upon the condition of the secreting epithelium. 
Collection and Preservation of the Urine. 

In all urinary examinations in which quantitative relations are to be 
studied it is necessary that a portion of the total 24-hour specimen be ex- 
amined. This should be thoroughly mixed and carefully measured. The 
composition of the different voidings is so variable that no definite idea 
regarding the elimination can be gained from a single specimen. 

^ See Lawrence (Am. Jour. Med. Sc, 191 2, CXLIV, 330) who shows that the amount of 
blood in a unit of time is of much more importance than the pressure. See, also, Leschke, 
Ztschr. f. khn. Med., i9i4,LXXXI, 14; Folinand Denis, Jour. Biol. Chem., 1915, XXII, 321; 
Davis, Jour. Urol., 1917, I, 113. 



l8o DIAGNOSTIC METHODS 

If a mere qualitative examination is to be made, a single specimen may be 
studied. In chronic nephritis, for instance, the morning urine may show 
points of interest as compared with that voided in the evening after a day's 
activity. In diabetes it may be desired to study the effect of a carbohydrate 
meal upon the sugar excretion. This may best be done by an examination of 
the urine passed three or four hours after such a meal. The microscopic 
examination is best made as soon as possible after voiding, but it is to be 
remembered that much variation may be noted in the sediment of the 
different voidings. 

In making the 24-hour collection, the patient is instructed to empty his 
bladder at a specified time, preferably at 7 a. m. This portion is thrown away 
and all urine passed from that time until the bladder is emptied at 7 A. M. the 
next day is saved. If one desires to separate the day and night urine, the 
voidings from 7 a. m. to 7 p. m. may be kept in one container and those from 
7 p. M. to 7 A. M. in a second vessel properly labeled. 

A thoroughly clean bottle of one-half to one gallon in capacity should be 
used as the container.^ This should be well corked after each addition of 
urine and kept in a cool place. As urine undergoes decomposition more or 
less readily, depending upon bacterial activity, some preservative should be 
added to prevent such processes. The writer is accustomed to advise the use 
of a slight excess of chloroform. This may be removed by heating the urine 
and will not then interfere with the later reactions. If not removed, its 
presence will lead to a pseudocarbohydrate reaction. Three or four drops of 
formalin may be added for each pint of urine. This is an efhcient agent, but 
it will lead to reactions simulating those for sugar and even albumin, and, 
moreover, will introduce a crystalHne compound of formalin and urea into 
the sediment as well as markedly interfering with the bile, urobilin^ and 
indican tests. Thymol may be added, but this may give a reaction similar to 
those for bile pigments, indican and albumin. Camphor, ^ chloral, and boracic 
acid have been used, but do not possess any virtues over the other preserva- 
tives mentioned. In any case, the worker should be on his guard in reporting 
abnormal findings without convincing himself that the reaction is not due 
to an added preservative. 

II. Physical Properties 
(i) Quantity. 

The amount of urine passed within 24 hours depends upon several factors 
and varies both for individuals and for different races of people. It is self- 
evident that under normal conditions, the amount of urine will vary with the 
quality and quantity of the substances to be excreted, the condition of the 
renal parenchyma, the pressure and rate of flow of the blood-current, the 
vasomotor disturbances, the stage of digestion, the loss of fluid in the per- 
spiration as influenced by the surrounding temperature, amount of exercise, 

^ See Folin and Denis, Arch. Int. Med., 1915, XVI, 195. 

2 See Hausmann, Deutsch. med. Wchnschr., 1913, XXXIX, 1685; Schmiz, Ibid., 1914, 
XL, 128. 

3 See Rosenbloom, New York Med. Jour., 1914, XCIX, 735. 



THE URINE l8l 

and extent of fluid intake; upon the weight of the subject, the sex, and 
age.i 

As a rule, the quantity of urine excreted varies between 1,200 and 1,500 
CO. (40 to 50 ounces), reaching a maximum two or three hours after a large 
fluid intake. Women excrete somewhat less than men, while children void 
relatively more than do adults, although the actual amount is less. In the 
adult we find the amount of urine is almost directly proportional to his 
weight, a normal large individual excreting nearer 1,800 than 1,500 c.c, the 
amount being about i c.c. per kilo and hour, while with a child the excretion 
is about 4 c.c. per kilo and hour. 

The physiologic limits of the urinary excretion are about 750 and 3,000 
c.c. In cases showing as high an output as 3,000 c.c. one is justified, per- 
haps, in assuming the presence of some pathologic condition. The kidneys 
are not easily deranged by the excess work put upon them in excreting large 
quantities of urine, so that we may find a secretion of many liters per day 
continuing for an extended period without endangering the normality of the 
excreting organ. 

Normally, the amount of urine excreted during the day by far exceeds that 
voided during the night, while the afternoon urine is usually more than that 
of the morning. We find, however, that in edematous conditions either of 
hepatic, cardiac, or renal origin, the night urine usually exceeds that of the 
day. This condition is known as nycturia. As it is, perhaps, more fre- 
quently associated with cardiac insufficiency, it may have a diagnostic 
importance in these cases. "^ 

Poljruria. 

By this is meant an excretion of an increased amount of urine. Just 
what amount of urine is to be considered as indicative of polyuria will depend 
much upon the habits of the patient as regards daily intake of fluid. As a 
rule, anything above 2,500 c.c. is at least suggestive of this condition. 

Just what factors are to be held accountable for the polyuria, is not always 
easy to decide in every case. An increased intake of fluid together with an 
increased general blood-pressure will cause both an increased local renal 
pressure and an increased blood-flow through the kidney. While such a 
polyuria rarely exists in cases of ordinary chronic or active renal hyperemia, 
we find under the influence of drugs that a very decided increase in the 
urinary output may occur. 

A polyuria is observed in the convalescent stages of acute nephritis, in 
both chronic parenchymatous and interstitial nephritis, and in amyloid de- 
generation of the kidney. This excretion, especially in the chronic inter- 
stitial type, may be one in which the total solids are normal or reduced, and 
is then known as hydruria. 

Diabetes mellitus is more frequently, perhaps, than other conditions 
associated with a polyuria. The quantity eliminated is dependent both upon 

^ See Addis and Watanabe, Jour. Biol. Chem., 1916, XXVII, 267; Haldane and Priest- 
ley, Jour. Physiol., 1916, L, 296; Priestley, Ibid., 304. 
^See Jespersen, Hospitalstid., 1916, LIX, 1229. 



102 DIAGNOSTIC METHODS 

the increased intake as a result of the polydipsia as well as upon the dehydrat- 
ing powers of the sugar. A certain relationship exists between the amount 
of fluid and the sugar, the polyuria being usually diminished by measures 
which decrease the amount of sugar excreted. This polyuria is not neces- 
sarily continuous and may alternate with periods showing a normal or 
subnormal amount of urine. 

In cases of diabetes insipidus we find the daily excretion of as much as 
50 liters or more of urine. According to Meyer, this polyuria is due to the 
attempt on the part of the kidneys to secrete sufhcient water to hold the 
solids in solution.^ 

In cases associated with abnormal accumulations of fluid, such as pleuritis, 
ascites, and general edema, a polyuria will exist at the time of absorption of 
the'exudates, owing to the presence of such large amounts of fluid in the 
blood-vessels.^ 

The so-called "epicritic polyuria" is frequently observed during convales- 
cence from acute febrile attacks. This is probably indicative of the elimina- 
tion of toxic products which have accumulated in the system during the 
progress of the disease. It is supposed to be of favorable import when occur- 
ring in a febrile condition, but it is to be recalled that this polyuria may be 
followed by a later oliguria which is of grave significance. As a rule, however, 
it may be said that as the case improves the urine is increased in amount. 

Polyuria may be observed in many nervous conditions, both functional 
and organic.^ The cause is probably some disturbance of the vasomotor 
apparatus as a result, perhaps, of irritation of the floor of the fourth ventricle, 
cerebellum, or cord. Hysteria, neurasthenia, epilepsy, and chorea are fre- 
quently associated with a polyuria. A paroxysmal polyuria in the course of a 
suspected nervous disease is more indicative of a functional derangement, while 
a continuous polyuria is more frequently associated with true organic disease. 

Oliguria. 

This is a condition characterized by the excretion of a diminished amount 
of urine, 800 c.c. being given as the lower normal point of the urinary output. 
Here, again, the absolute figure must depend upon the patient and upon his 
customary excretion. A single examination is not sufficient to decide 
whether or not an oliguria exists. 

^ See Lewis and Matthews (Trans. Chic. Path. Soc, 1913, IX, 16) who show the close 
relation of hypersecretion of the pars intermedia of the hypophysis to diabetes insipidus. 
See Socin, Ztschr. f., khn. Med., 1913, LXXVIII, 294; also, Forschbach, Ibid., 1913, 
LXXVII, 153; Goldzieher, Verhandl. d. deutsch. path. Gesellsch., 1913, XVI, 272; Ber- 
blinger, Ibid., 281; Simmonds, Miinch. med. Wchnschr., 1914, LXI, 180; Hohlweg, Ibid., 
927; Fitz, Arch. Int. Med., 1914, XIV, 706; Mathews, Ibid., 1914, XV, 451; Krikortz, 
Hygiea, 1915, LXXVII, 49; Motzfeldt, Boston Med. & Surg. Jour., 1916, CLXXIV, 644; 
Jour. Exper. Med., 191 7, XXV, 153; Rosenbloom and Price, Am. Jour. Dis. Child., 1916, 
XII, 53; Newmark, Arch. Int. Med., 1917, XIX, 55c; Christie and Stewart, Ibid., XX, 
10; Oehme, Deutsch. Arch. f. klin. Med., 1918, CXXVII, 261; Leschke, Ztschr. f. klin. 
Med., 1919, LXXXVII, 201; Kennaway and Mottram, Quart. Jour. Med., 1919, XII, 
225; Maranon and Gutierrez, Sig. Med., 1919, LXVI, 809; Lereboullet, Progres. Med., 
1919, XXXIV, 363. 

2 See Baehr, Deutsch. Arch. f. klin. Med., 1913, CIX, 417; also, Leuret, Jour, de med. 
de Bordeaux, 1913, LXXXIV, 669. 

2 See Pirondini, Jour. d'Urol., 1914; V, 461; Mathieu, Bull. Acad, de Med., 1915, 
LXXIII, 208. 



THE URINE 183 

This condition is found, perhaps, most frequently in cases of broken 
compensation of the heart, where the blood-pressure is markedly diminished. 
It is present whether the cardiac incompetency be primary or secondary to 
hepatic, renal, or pulmonary lesions. 

Oliguria is noted in practically all acute febrile disorders, especially in 
typhoid fever. This is due, probably, to a combination of cardiac weakness 
with the increased loss of water by the skin and lungs. Moreover, we may 
have, in such states, a retention of fluid along with a direct contraction of the 
renal vessels. 

Acute nephritis as well as chronic parenchymatous nephritis are asso- 
ciated with a more or less extensive oliguria. This condition is probably 
referable to diminished functional activity of the glandular elements as well 
as to increased resistance within the tubules. A bilateral diffuse lesion is 
always necessary to cause much oliguria, as the sound kidney, if the trouble 
be unilateral, will take on vicarious activity. The more acute the condition, 
the greater the degree of oliguria. 

Oliguria may also occur following the administration of an anesthetic, 
in connection with • eclampsia, hysteria, or epilepsy, after the loss of large 
quantities of fluid by hemorrhage, diarrhea, or vomiting, in cases of portal 
obstruction as seen in acute yellow atrophy or hepatic cirrhosis, or in cases in 
which pressure is exerted upon the vascular system, especially the vena cava, 
by tumors.^ 

Anuria. 

This oliguria may, in almost any case, proceed to complete anuria, which 
may or may not be of vital significance. Cases of anuria do occur without 
any preceding oliguria, as shown in acute nephritis and in some cases of 
hysteria. Anuria, per se, cannot be held responsible, however, for the 
uremic symptoms so frequently associated with it, as it may persist for many 
days, 19 in a case of Adams, without any uremic signs. 

Anuria may be due to obstructive, reflex, renal, and prerenal causes. We 
may have an occlusion of the urinary passages^ on one side and a reflex closure 
on the other. Tumors, prostatic hypertrophy, and toxic and nervous bladder 
disturbances may lead to a great degree of oliguria amounting, almost, to anuria. 

The so-called prerenal causes of anuria include scarlet fever, which may 
lead to a severe nephritis, phosphorus poisoning, action of ether and chloro- 
form, collapse, ureteral and urethral calculus, and cholera. 

(2) Appearance. 

Freshly voided urine should be clear and transparent. Only the faintest 
trace of any turbidity should be normally present, except soon after a meal 
rich in vegetable food, when a distinct turbidity may be noticed due to the 
precipitation of the phosphates in the alkaline urine. 

^ See Cohn, Berl. klin. Wchnschr., 1915, LII, 208; von Szollosy, Ztschr. f. exper. Path, 
u. Therap., 1915, XVII, 243, calls attention to the importance of opsiuria (a retardation of 
the urinary output) from the diagnostic standpoint. 

2 See MacNider (Jour. Med. Research, 191 2, XXVI, 79) for a discussion of the relation 
of swelling of the tubular epithelium to the urinary output. Also, Cecil, Jour. A. M. A., 
191 7, LXVIII, 440. 



184 DIAGNOSTIC METHODS 

When allowed to stand for a short time, a light cloud is noted which 
gradually settles to the bottom of the container in the form of the so-called 
*' nubecula." This contains a few small granular cells and a few epithelial 
cells and is composed largely of mucus. 

On standing for a sornewhat longer period, as for instance over night, 
at the ordinary temperature, distinct crystals of uric acid may separate and 
appear in the sediment. If the temperature of the urine is allowed to fall to 
a considerable extent during this period, a somewhat more marked turbidity 
will be produced owing to the precipitation of the acid urates. . This sediment 
is particularly noticeable if a highly acid urine becomes very cold. 

If kept for a longer period at room temperature, or a shorter period during 
the warmer months, a diffuse cloudiness will appear, due to the precipitation 
of the phosphates, owing to the lessened acidity or abnormal alkalinity of the 
urine. This alkalinity is due to the decomposition of the urea into am- 
monium carbonate. The crystals in this alkaline urine will be triple 
phosphates, calcium phosphates, ammonium urate, and calcium carbonate. 

Even before the urine becomes alkaline, a diffuse cloudiness may be 
present, due to the development of numerous saprophytic bacteria. This 
bacterial cloud is removed only with the greatest difficulty, as filtration of the 
urine has practically no effect upon it. Frequently, the addition of lead 
acetate to the urine will produce a voluminous precipitate, which may carry 
down the bacteria and permit of their filtration. This procedure is, however, 
not to be recommended, as other substances, if present in small amounts, may 
be carried down and thus escape detection. 

If the urine is cloudy when freshly voided, the turbidity may be the result 
of the precipitation of phosphates through the alkalinity or it may indicate the 
presence of an organized sediment, such as casts, epithelial cells, blood, and 
pus. 

The normal urine shows but very little viscosity, differing little from 
ordinary water in this respect.^ In certain conditions we find a marked degree 
of viscidity, which becomes especially apparent on attempting to filter the 
urine. In cases of chronic cystitis the excretion of a large amount of mucus 
may make the urine ropy and gelatinous. This increased viscosity may also 
be seen in cases of pyuria associated with decomposition.^ 

(3) Color. 

The color of the urine varies normally between various shades of yellow, 
the depth of color depending upon the concentration or specific gravity of 
the specimen. While the color is usually much paler in the urines of low 
specific gravity and very dark in those of high density, we find in diabetes 
mellitus a very pale urine with a high specific gravity. In cases of anemia 
the urine is always paler than normal, but in pernicious anemia the urine 
is highly colored owing to the marked destruction of the erythrocytes. 

As a rule, it may be said that an acid urine is more highly colored than 

^ See Posner, Berl. klin. Wchnschr., 1915, LII, 1106. 

2 See Steensma (Nederl. Tijdschr. v. Geneesk., 1914, LVIII, 24), who reports fluores- 
cence in certain types of urines. 



r 



THE URINE 185 

an alkaline one, although many exceptions to this rule occur. There seems 
to be some difference between the urines passed at different periods of the 
day; thus the urine of the day is usually a distinct amber, while that of the 
night may take on a greenish tinge. To what this color in the latter case is 
due is at present unsettled. Several color scales have been introduced, such 
as those of Neubauer and Vogel and of Radde, but these are not sufficiently 
extensive to take in pathologic variations where they would be most impor- 
tant. As a rule, it is sufficient to divide the colors of the urine into those of the 
spectrum, making allowance for light, medium, and dark shades of each color. 
The pigments causing the normal and abnormal colorations of the urine 
will be discussed in detail in a later section. At this point the writer would 
say that normally these pigments are urochrome, uroerythrin, and urobihn, 
while the various conjugated glycuronic and sulphuric acids, blood pigments, 
biliary pigments, melanin, etc., are found in pathologic conditions. 

Pathologic Colorations. 

Deviating from the rule that the higher the specific gravity the more in- 
tense the color, diabetes mellitus shows an extremely light color with a 
high specific gravity. Owing to the lack of pigment, in chlorosis we find 
a very pale urine. Chronic interstitial nephritis and amyloid degeneration 
of the kidneys are associated with an extremely pale urine. 

In febrile conditions the coloration may range from an orange-red to a 
distinctly red tint, owing to the increase in the amount of urobilin. This 
deep color is especially noticeable in cases of severe pneumonia.^ This red- 
dish urine may also be due to an increase in the amount of uroerythrin, which 
is responsible for the deep color of the urate sediment so frequent in the con- 
centrated urines of febrile conditions, circulatory disturbance of the liver, and 
in cases associated with profuse perspiration. 

In cases of jaundice the urine may vary from a dark yellow or green to a 
brown or black, depending upon the concentration of the urine, the amount 
of bile pigments, and upon certain chemical activity which occurs in such 
urine. Not only will the color of the urine be deeper, but the foam which 
appears on shaking the specimen will take on a distinct yellowish-brown tint. 
If this biliary urine be allowed to stand in the cold for some time, crystals 
of bilirubin may separate out and be seen in the sediment. 

Urine which contains blood may have a violet shimmer, may appear 
smoky, blood red, brownish-black, or even deep black in color. These varia- 
tions depend both upon the amount and kind of pigment present. Hemo- 
globin gives a more reddish tint to the urine, while methemoglobin produces a 
brownish shade. Such urine is always cloudy, owing to the admixture of 
corpuscles and other organic material. The blood found in the urine may 
arise from any point in the genitourinary tract or may be of systemic origin. 
In the latter case conditions which give rise to hemolysis will cause the appear- 
ance of hemoglobin in the urine. 

The condition of chyluria is characterized by the presence of large numbers 
of highly refractile globules of fat along with many morphological constitu- 

1 See Hildebrandt, Ztschr. f. klin. Med., 191 1, LXXIII, 189. 



1 86 DIAGNOSTIC METHODS 

ents. This gives rise to the appearance of a milky urine and is especially 
characteristic of infection with the filaria. It is not infrequent to find, in cer- 
tain cases of hysteria, a specimen of milky urine, owing to the fact that the pa- 
tient has added milk to the urine before sending it to be examined. The pres- 
ence of a large quantity of pus will also give the urine a milky appearance. 

The urine of patients suffering with melanotic tumors may be perfectly 
clear when freshly voided, but becomes black or dark brown on exposure to 
the air. This reaction is due to the transformation of the pigment melanogen 
to melanin, and may be hastened by the addition of oxidizing agents to the 
urine. This darkening of the urine extends characteristically from above 
downward. 

The condition known as alkaptonuria, which is characterized by the ex- 
cretion of homogentisic and uroleucic acids, gives rise to the passage of a urine 
which is brownish-black in color and may be syrupy in consistency. This color 
is not always evident in the fresh specimen, but appears soon after being voided. 

In cases of peritonitis, suppuration anywhere in the system, gangrene, 
and marked intestinal putrefaction, the urine is frequently dark colored owing 
to the passage of certain aromatic products of decomposition, either indican 
or various derivatives of phenol. The coloration in these cases may vary from 
a dark brown or greenish-black to a distinct blue. These urines differ from 
those containing melanin in the fact that ferric chlorid does not blacken 
the urine as it does in the presence of melanin. This urine may contain a 
distinct amount of indigo, although the substance present is usually a different 
oxidation product. If indigo be present, a bluish-black scum will frequently 
rise to the surface of the specimen. 
Medicinal Coloration. 

After the use of carbolic acid either internally or externally, guaiacol, 
creosote, resorcin, naphthalin, salol, and various tar preparations the urine 
may vary from a dark brown to a black color. This coloration is due to the 
excretion of hydroquinon and of pyrocatechin, and may be evident only on 
allowing the urine to stand for some time. The urine containing pyrocate- 
chin may reduce alkaline copper solutions, but will not affect such bismuth 
preparations. While a dark brown or black coloration of the urine may be 
found in cases of hemorrhage, melanosis, malaria, alkaptonuria, ochronosis, 
and chronic tuberculosis, one should be on his guard, as the medication of the 
case may be responsible for such coloration. 

Methylene blue will color the urine a greenish to deep blue shade, which 
may last for several days. Usually within an hour after this drug is taken the 
urine may show a faint tinge of green which may be more clearly brought 
out by acidifying with acetic acid and warming. 

The use of the hypnotics, trional, sulphonal, and tetronal, frequently 
gives rise to the voiding of a urine which has a deep red-wine color, due to the 
presence of hematoporphyrin. Pyramidon produces a urine of rose-red color, 
the pigment of which is soluble in ether, chloroform, and amyl alcohol. Anti- 
pyrin and purgatin both produce distinctly red urines. Chrysarobin, senna, 
rhubarb, cascara, and santonin produce a golden-yellow urine which becomes 



THE URINE 187 

red in the presence of alkali. This coloration is due to the excretion of 
chrysophanic acid. According to Gorup-Besanez, the pigment of beets, 
huckleberries, blackberries, etc., may under certain conditions be excreted 
in the urine and color it the corresponding shades. 

(4) Odor. 

The normal urine usually has a distinct aromatic odor which very much 
resembles that of beef broth. This odor is due to the presence of certain 
volatile acids and is more marked in urines of high concentration.^ If the 
urine undergoes decomposition either within the bladder or on standing, a 
so-called "urinous odor^' appears which is due to the decomposition of protein 
material. This odor is very markedly ammoniacal. Should such an odor 
appear in the freshly voided specimen, it is evidence of marked cystitis. 

Abnormal decomposition of the urine, as evidenced by changes in the odor, 
may be found in conditions associated with decomposition of pus and may 
be due to the presence of hydrogen sulphid along with the ammonia. This 
condition may be observed in cases of perforation of an abscess into the urin- 
ary tract, in which case the urine may have a distinctly fecal odor if the intes- 
tine be involved, while in carcinoma of the bladder this repulsive odor of the 
urine may also be noticed. 

A distinct fruity odor is often present in cases of diabetes melHtus, in 
many febrile conditions, and in some stomach and intestinal troubles, which 
may be directly traceable to the presence of acetone. 

Certain medicaments, such as oil of turpentine, give rise to a distinct 
odor of violets in the urine. Menthol causes an odor of peppermint, while 
cubebs, copaiba, sandal-wood oil, tolu, and saffron produce a peculiar spicy 
odor. Valerian and asafetida are excreted as such in the urine and produce 
their characteristic odor. Certain foods, such as meat, bouillon, and coffee, 
produce a slight odor of the urine, while asparagus gives a peculiar char- 
acteristic odor due to the presence of methyl mercaptan. 

(5) Reaction. 

The normal urine has an acid reaction. According to the views formerly 
held, this acidity was directly due to the presence of acid salts, especially to 
sodium dihydrogen phosphate (NaH2P04), and not to the presence of any 
free acid. 

The recent work of Folin has shown that the phosphates in the clear urine 
are all of the monobasic (diacid) type. His figures indicate that the acidity 
of normal clear urines is ordinarily greater than the acidity of all the phos- 
phates present and that the excess must be due to free organic acids. For 
this reason the methods of Freund and of Lieblein for the determination of the 
acidity of the urine must be given up. To quote from Folin i^ ''The current 
attractive, and in a measure plausible, belief that the acidity of urine is regu- 
lated by variations in the relative proportion of the two forms of 'acid 

1 Dehn and Hartman (Jour. Am. Chem. Soc, 1914, XXXVI, 2118 and 2136) believe the 
odor of urine to be due to a peculiar substance which they call "urinod." See, also, Hart- 
man, Arch. Int. Med., 1915, XVI, 98. 

2 Am. Jour. Physiol., 1905, XIII, 45. 



1 88 DIAGNOSTIC METHODS 

phosphates' is, therefore, erroneous. If urine does at no time contain com- 
paratively strong acids in the free form, the reason is in part the variability of 
the ammonia formation and in part the presence of salts of organic acids. In 
a mixture of salts containing an excess of acids it is the weakest which will re- 
main uncombined and the strongest organic acids will, therefore, exist as 
salts; but if the total amount of acidity becomes abnormally great, the 
quality (the strength) of the free acids may change." 

From the standpoint of physical chemistry the acidity of the urine, as of 
all other acid solutions, should represent the absolute number of dissociated 
hydrogen ions in a definite quantity of the urine. ^ We are, therefore,'face to 
face with the same problem confronting us in the examination of the alkalinity 
of the blood. In the case of the urine the question of indicator to be used in 
the titration test is a matter of great moment, as no two indicators will give 
the same degree of acidity. The one naturally to be selected would be that 
which will react to every possible substance of an acid nature. 

If we use the methods of physical chemistry we find, according to Hober,^ 
that the urine is only about 30 times as acid as is distilled water and only 
about one ten-thousandth as acid as the titration figures would indicate. 
Such being the case, we must either entirely revise our figures for the acidity 
of the urine or employ methods which can be more easily carried out by the 
general worker than can those of physical chemistry.^ 

Folin has, therefore, introduced a method which uses direct titration 
of the urine and employs phenolphthalein as an indicator. This indicator 
reacts to all bodies of an acid nature, but cannot overcome certain difficulties 
which are in the way of direct titration. These obstacles are (i) the occur- 
rence of calcium in the urine in the presence of the monobasic phosphates, and 
(2) the presence of ammonium salts. He has found that the addition of 
potassium oxalate to the urine will do away with these difiiculties by holding 
in solution both the di- and tri-calcium phosphates and by preventing the 
dissociation of the ammonium compounds. 

Folin' s Method. 
Total Acidity. 

Twenty-five c.c. of urine are treated with 15 to 20 grams of powdered 
potassium oxalate and one or two drops of a i per cent, alcoholic solution of 
phenol-phthalein. The mixture is shaken rapidly for one or two minutes 
and titrated^t once with a tenth-normal sodium hydrate solution until a 
faint, distinct, permanent pink color is obtained. It is advisable to shake 

^ For a consideration of methods for determining the H-ion concentration see Chapter 
on Blood. The Ph values for urine are, of course, subject to wide variation under different 
influences but the usual figures run from 5.98 as reported by Henderson and Palmer (Jour. 
Biol. Chem., 191 2, XIII, 393 and 191 4, XVII, 305) to 6.64 as shown by Blatherwick, 
x\rch. Int. Med., 1914, XIV, 409. 

2 Beitr. zur chem. Physiol, u. Path., 1903, III, 525. 

3 See Henderson and Palmer, Jour. Biol. Chem., 1913, XIII, 393; Ibid., 1913, XIV, 81; 
Arch. Int. Med., 1913, XII, 153; also, Henderson, Science, 1913, XXXVII, 389; Henderson, 
Palmer and Newburgh, Jour. Exper. Pharmacol., 1914, V, 449; Henderson and Palmer, Jour. 
Biol. Chem., 1914, XVII, 305; Ibid., 1915, XXI, 37; Ibid., 57; Arch. Int. Med., 1915, XVI, 
109; Host, Ztschr., f. klin. Med., 1915, LXXXI, 266; Van Slyke and Palmer, Proc. Soc. 
Exper. Biol, and Med., 1919; XVI, 140. 



THE URINE 189 

the flask during the titration so as to prolong the effects of the potassium 
oxalate. The acidity is expressed in terms of the amount of tenth-normal 
sodium hydrate solution necessary for neutralization of the 24-hour amount of 
urine. This is expressed as T, which is, on an average, 617. 

The studies of Fitz and Van Syke on acid excretion were undertaken 
to ascertain whether a quantitative relationship could be discovered between 
the alkaline reserve of the blood plasma, as measured by its combining power 
for CO2, and the rate of acid excretion by the kidneys. It has been known for 
some time that entrance of acid into the circulation immediately reduces 
the blood bicarbonate and is accompanied by an increased rate of acid and 
ammonia excretion in the urine. In this work the plasma bicarbonate is esti- 
mated by the CO2 combining power method of Van Slyke; the ammonia by 
the permutit method of Folin and Bell (as discussed on page 241) ; and the 
acid titratable with phenolphthalein as an indicator by the method of Folin 
given above. This latter was selected because the acid titratable with phenol- 
phthalein approaches zero in human urine when the height of the plasma 
bicarbonate is at its maximum normal of about 80 volumes per cent., under 
which conditions ammonia excretion also approaches zero. These workers 
present their results in the form of an equation, comparable to those of Am- 
bard and of McLean for the urea and chlorid relations of the blood and urine 
(as discussed later), which permits of the determination of the relationship 
of this excretion to the alkaline reserve of the body as measured by the CO2 
combining power of the blood plasma. The original formula of Fitz and Van 
Slyke for this relationship is as follows: 



wVc, h 



Plasma CO2 Capacity — 80 — \/^ V C, in which D represents the rate 

of excretion of o.i N ammonia plus o.i N titratable acid per 24 hours; C 
the O.I N ammonia plus 0.1 N acid per liter of urine, and W the body weight. 
The index may be determined from analysis of the urine passed in 24 hours 
or from the amount excreted in i or 2 hours, multipHed to bring the data to 
a 24 hours basis. From the work of Barnett, it is evident that the average 
values for the fourth root of C is 5. Hence the above formula may be sim- 
plified as follows without impairing the general accuracy: 



w = 



Plasma CO2 Capacity = 80 — 5 Vtt? = Index of Acid Excretion. 

As Stillman, Van Slyke, Cullen and Fitz show, the urine index in very severe 
acidosis is less accurate that the alveolar air in indicating the alkaline reserve. 
With a plasma CO2 of 25 per cent., which corresponds to a urine index of 55, 
the index may be 65, indicating 15 per cent, plasma CO2, which is fatal in 
their experience, or it may be 45, indicating 35 per cent, plasma CO2, which, 
though a pronounced acidosis is so well above the danger limit that signs of 
coma are usually absent. The alveolar carbon dioxide method appears the 
more accurate in measuring the more severe stages of diabetic acidosis,while 
the index of acid excretion is the more accurate in measuring the more com- 



I go DIAGNOSTIC METHODS 

mon intermediate stages. The normal values for this index range from 3 to 
27. Anything above 27 indicates an acidosis, which usually becomes crit- 
ical if it approaches 100 c.c. per kilo. It is to be remarked that the results 
by this method do not agree within approximately 10 per cent., so that de- 
viations of this amount either way must be allowed for.^ 

Free Mineral and Organic Acidity. 

Determine the amount of total phosphates present by titration with 
uranium nitrate solution as described later. Seven and one one-hundredth 
mg. of P2O5 have an acidimetric value equal to i c.c. of tenth-normal acid. 
The total acidimetric value of the phosphates of the 24-hour urine may he 
easily determined with the help of this factor, by converting the amount of 
phosphates into terms of N/io acid. 

From the total acidity (T) subtract the acidimetric value of the phosphates 
(P). The remainder is the acidity due to uncombined organic acids, and 
the difference, that obtained from calculating all the phosphoric acid as di- 
acid phosphate, is the free mineral acidity. For all ordinary studies of the 
acidity of the urine the direct titration of the total acidity and of the phos- 
phates gives the necessary information. ^ The excess of the total acidity 
above that calculated from the phosphates gives the total free acids present. 
If the acidity calculated from the total phosphates is greater than the titrated 
acidity, then there are practically no free organic acids present, and the ti- 
trated acidity represents the amount of phosphates present in the diacid form 
(Folin). 

While the acidity of the urine is best determined and expressed as outlined 
above, it seems wise to the writer to retain the same style of expression for the 
acidity as used in stomach analysis. With this nomenclature one would state 
the acidity of the urine in degrees, that is, the amount of tenth-normal sodium 
hydrate necessary to neutralize 100 c.c. Under normal conditions this will 
vary from 35 to 45°. It may be increased by a diet rich in meat, while it is 
decreased by a vegetable diet. There are many acids produced in the oxida- 
tion of protein, among which we find sulphuric, phosphoric, uric, and the 
oxyaromatic acids. Ordinarily, these play an indirect part in the acidity of 
the urine, although this phosphoric acid may exist in part as the dihydrogen 
phosphate and in consequence increase the acidity of the urine. The regula- 
tion of the metabolism is such that an increase of the acids produced in the 
system or taken into it from without is neutralized by an increased formation 
of ammonia, the salts appearing in the urine as the ammonium salts which do 
not, of course, increase the acidity of this fluid. This is the basis upon which 
one estimates the amount of ammonia in following a condition of acidosis.^ 

^Fitz and Van Slyke, Jour. Biol. Chem., 1917, XXX, 389; Stillman, Van Slyke, Cullen, 
and Fitz, Ibid., 405; Barnett, Ibid., 1918, XXXIII, 267; Van Slyke, Ibid., 271. 

^See Walpole (Biochem. Jour., 1914, VIII, 628) for a discussion of various new 
indicators. 

^ Fischer (Nephritis: An Experimental and Critical Study of Its Nature, Cause and the 
Principles of Its Relief, New York, 191 2) believes that the abnormal production or accumu- 
lation of acid in the cells of the kidney and the action of this acid on the colloidal structures 
of the kidney are accountable for all the changes which characterize nephritis. See, also, 
Hirschfelder, Jour. A. M. A., 1916, LXVII, 1891. 



THE URINE 191 

The reaction of the urine varies at different times of the day. The acidity- 
appears to be highest in the morning before breakfast and is diminished after 
a meal, due to the secretion of hydrochloric acid into the stomach. The re- 
action of the urine may even be alkaline for a period of two or four hours after 
each meal, in which case the urine will be turbid from the precipitation of 
phosphates. This reaction of the urine following meals is known as the 
"alkaline tide" of the urine. Between meals the acidity of the urine will 
gradually increase until the next meal is taken. 

The reaction of the urine is modified to a great extent by the use of drugs. ^ 
Thus, alkalies, such as carbonate and bicarbonate of sodium, will render 
the urine alkaline if taken between meals, while if taken just preceding a meal 
they will be neutralized by the gastric juice. All organic acids of the fatty 
series are oxidized in the system to carbonic acid and combine with bases 
forming basic salts which render the urine alkaline or less acid, providing 
these acids are not taken above the point of tolerance, as the writer has shown 
that large doses of such acids as citric acid' will increase the acidity of the 
urine. The mineral and aromatic organic acids will, however, practically 
always increase the acidity. 

In many pathologic conditions we find the reaction of the urine variable.^ 
Abnormal gastric activity may be either associated with an increase or a 
decrease in the acidity of the urine, depending upon a condition of hypo- or 
hyperacidity of the gastric juice. The rapid absorption of a transudate or 
exudate will lead to the excretion of an alkaline urine f ron the presence of an 
increased amount of alkaline salts. An alkaline urine is. not infrequently 
seen after intestinal hemorrhage, in certain cases of pneumonia, typhoid fever, 
chronic nephritis, and in cases in which exudates from the urinary tracts have 
become mixed with the urine. In certain cases of nervous diseases and in 
some cases of anemia we may also find an alkaline urine. The urine in all 
of the above cases will show, if tested by litmus-paper, an alkaline reaction in 
which thie blmng of the red litmus-paper is permanent. This condition is 
known sls fixed alkalinity and is quite distinct from the following type. 

In cases of decomposition of the urine within the urinary tract, through 
the influence of bacteria, the urea is decomposed into ammonium carbamate 
and carbonate. The alkaline reaction of the urine in such cases will be 
shown by a blue color of the red litmus-paper either held above it or placed 
in it, the blue color disappearing when the paper is dried. This condition 
is known as volatile alkalinity. If the urine shows this volatile alkalinity 
on being voided, the finding is significant of trouble somewhere along the 
urinary tract, especially within the bladder.^ 

Benedict has recently introduced the term "acid unit" into the study of 
the urinary output. One c.c. of urine with an acidity of i degree is equivalent 
to 100 acid units. In other words, one may determine the acid units in the 

1 See de Jager (Biochem. Ztschr., 191 2, XXXVIII, 294), who shows that magnesium sul- 
•phate increases the acidity, while sodium sulphate diminishes it. 

2 See Newburgh, Palmer and Henderson, Arch. Int. Med., 1913,'XII, 146; also, Pertik 
(Virchow's Arch. f. path. Anat., 1913, CCXIII, 465) who finds a decreased acidity in tuber- 
culosis; Labbe and Vitry, Presse Med., 1914, XXII, 437. 

3 See Saidman, C. R. soc. biol. de Paris, 1916, LXIX, 780. 



192 DIAGNOSTIC METHODS 

24-hour specimen by multiplying the number of cubic centimeters of the 
urine by the degrees of acidity. This is normally about 40,000. 

(6) Specific Gravity. 

The specific gravity of the normal urine ranges between 1,015 ^^d 1,025, 
with an average of 1,020. This specific gravity will depend, of course, upon 
the amount of fluid intake, the quantity of the 24-hour specimen of urine, the 
degree of tissue activity, and the condition of the secreting organs. The 
intake of a large volume of water may reduce the specific gravity of the urine 
to a very low figure and, correspondingly, a small intake may lead to a urine 
of high gravity. We find, therefore, that perfectly normal urines may show 
specific gravities ranging from 1,010 to 1,030, with pathologic variations from 
1,002 as high as 1,060 or more.' 

In general routine work it is essential that the specific gravity of the 
24-hour specimen be determined. Except in unusual cases, absolutely noth- 
ing of diagnostic value may be learned by the determination of the specific 
gravity of a single voiding of urine. The variations at different times of the 
day, under the influence of food, digestion, activity of the skin and lungs, 
and exercise, may be so great that apparent pathologic figures may be ob- 
tained from a single specimen. Such variations are overcome for the most 
part in the 24-hour specimen unless pathologic conditions are present to keep 
up such variations. In some cases, especially in chronic diffuse nephritis, 
the morning specimen of urine almost invariably has a lower specific gravity 
than that of the other periods of the day. For this reason one may determine 
the specific gravity of a single specimen of such urines. 

It is of especial importance that the total amount of urine in the 24-hour 
specimen be taken into consideration in judging of the value of a specific 
gravity. Thus in chronic interstitial nephritis we may find a large volume 
of urine with a low specific gravity, while in diabetes mellitus an even higher 
volume of urine may be present, showing a vtry high specific gravity. 
Technic. 

The most accurate method of determining the specific gravity is, of 
course, the use of the pycnometer. The principle of this method is the de- 
termination of the weight of a definite volume of urine as compared with 
that of the same volume of distilled water under the same conditions of 
temperature and atmospheric pressure. This method will be discussed in 
the section on Blood, to which the reader is referred. 

The clinical method of estimating this factor is a distinctly areometric 
one. The principle of this method is that a body immersed in a fluid will 
displace an amount of fluid equivalent to the loss in its own weight. By 
the use of instruments known as hydrometers or in the case of the urine as 
urinometers, this displacement is measured by immersing the hydrometer 
in the fluid and observing the point to which this instrument sinks. The 
stem of the hydrometer is graduated in divisions from 1,000 to 1,060 by 
differences of 1°, the t,ooo point being that to which the instrument sinks 

^ See Frey, Deutsch, Arch. klin. Med., 191 2, CVI, 347. 



THE URINE 



193 



when immersed in distilled water at the temperature to which the instru- 
ment is calibrated. Any variation in the density of the solution in which 
this hydrometer is immersed will be evident by the depth to which it sinks, 
the more concentrated the solution the less will the instrument sink. 

The vessel in which the urine is poured should be cylindrical in shape, 
with parallel sides and wide base and sufficiently tall to permit of the complete 
sinking of the hydrometer. The forms of this cylinder 
with fluted sides are perhaps more desirable than the 
plain cylinder, as the bulb does not tend to stick to the 
sides of the vessel so readily. 

The vessel is filled about four-fifths full of urine, any 
foam being removed by the use of filter-paper. The hy- 
drometer is placed in the urine with a twisting motion and 
allowed to come to rest. The depth to which the stem 
is immersed is then read off by observing the mark which 
coincides with the lower meniscus of the urine as seen from 
below. The worker should never attempt to read the 
specific gravity from above as a slight meniscus interferes 
with the accuracy of his reading. The worker must be 
absolutely sure that the urinometer neither rests upon the 
bottom of the cylinder nor touches the sides, but should 
see that it floats perfectly free in the urine. 

The temperature at which the reading is taken is a 
matter of some moment as some of these instruments are 
graduated at i5°C. The ordinary model, as made by 
Squibb, is graduated at 25^0., which is, perhaps, niore 
nearly the working temperature of the room. A variation 
of 3° in temperature between that of the room and that at 
which the instrument is calibrated, will give a difference of 
1° of specific gravity; that is, a difference in the fourth 

place of the specific gravity. In ordinary clinical work . 

corrections for variations in temperature are usually un- ometer and cylin- 
necessary, as variations of two or three points in the fourth ^^^- (Hawk.) 
place of the specific gravity are of absolutely no impor- 
tance, as such changes might be attributed to chemical variations on stand- 
ing, even though the most accurate methods of estimating the specific gravity 
were used. 

If the quantity of urine be very small, it may be diluted with distilled 
water, so that the measuring cylinder may contain enough material to permit 
of a density estimation. The specific gravity of this diluted urine is then 
determined as above and the last two figures of the specific gravity are 
multiplied by the degree of dilution. 




Rough Estimate of Total Solids. 

As the degree of specific gravity is directly proportional to the amount 
of solids contained in the urine, one may roughly judge of the total solids 



13 



194 DIAGNOSTIC METHODS 

by a simple calculation as follows: If the last two figures of the specific 
gravity be multiplied by 2.33 (Haeser's coefficient), the result will be the 
approximate number of grams of total solids in every 1,000 ex. of urine. 
Knowing the quantity of urine passed in the 24 hours, a simple calculation will 
yield the total 24-hour excretion of solids. Long uses the coefficient 2.6. 
Instead of the above figure one may multiply the last two figures of the specific 
gravity by i.i (Haines' coefficient) and obtain the number of grains of solids 
in each fluidounce of the urine. This figure when multiplied by the total 
number of ounces of the 24-hour specimen will yield the excretion of soHds 
in grains. This latter method has some advantage for the older practitioner 
who has not accustomed himself to the use of the metric system. 

It is to be said that neither one of the above methods can give anything 
but approximate results and in pathologic urines are absolutely unreliable. 
Highly albuminous urines invariably show a reduced specific gravity, while 
a high sugar content is associated with an increase in the density. In either 
one of these cases a calculation of the total solids by the above method will 
yield inexact figures. If the worker desires to know the exact amount of 
total solids in the 24-hour specimen, and this is sometimes advisable, recourse 
should be had to more exact methods of determination which will be discussed 
later. 

The specific gravity of a specimen of urine varies, of course, with the 
amount of total solids.^ Normally, these range from 60 to 70 grams with a 
24-hour excretion of 1,500 c.c. of urine. The urea usually constitutes about 
one-half of the total solids. As the normal percentage of urea is approxi- 
mately two with a specific gravity of 1,020, the writer has been struck with 
the usual close relationship of the percentage of the urea to the specific 
gravity. In watching this point in over 2,000 examinations of urine within 
the last year, the writer has observed that the percentage of urea will prac- 
tically parallel the last two figures of the specific gravity; in other words a 
specific gravity of 1,015, for instance, will normally be associated with a urea 
content of 1.5 per cent. This statement is true only in those cases which 
contain neither albumin nor sugar. 

As a rule, it may be said that the specific gravity of the urine is inversely 
proportional to the amount of fluid eliminated. It will, therefore, be evident 
that conditions leading to an oliguria will produce a high specific gravity, 
while those causing polyuria will give a low specific gravity. This state- 
ment must be modified when considering certain pathologic conditions, as 
we may find a diminished amount of urine with low specific gravity as in 
chronic nephritis in which the salts are diminished, although the organic 
albuminous bodies are much increased; while in diabetes mellitus we have an 
abundant urine of high specific gravity. 

The specific gravity of the urine is of more or less importance in judging 
of the activity of the kidneys. In acute nephritis we find a urine of high 
specific gravity, while in the chronic types of renal disease the specific gravity 
is low owing to the diminution of the salts. It is to be noted in this connec- 

1 See Jacob, Deutsch. Arch. f. klin. Med., 1913, CX, i. 



THE URINE 195 

tion that in the case of so-called "functional" albuminuria, the specific 
gravity of the urine is above the normal figures. A marked reduction in the 
specific gravity of any case of nephritis is of dangerous import. 

(7) Optical Activity. 

According to Haas,^ the normal urine is slightly levorotatory, ranging 
from o.oi to 0.18. This optical activity is due to traces of the conjugated 
glycuronic acids which will be discussed later. 

An increase in this levorotatory power is observed due to the presence 
of increased amounts of glycuronic acid, /3-oxybutyric acid, albumin (in 
amounts over one-half part per thousand), and levulose. Dextrorotatory 
urines depend upon the presence of glucose, maltose, and lactose, while the 
presence of pentose usually gives rise to an optically inactive urine or one 
at least showing only a slight degree of dextrorotation. 

This optical activity under the influence of pathologic products will be 
discussed in the section on Carbohydrates, to which the reader is referred. 

III. Chemical Properties 

(A) Normal Composition. 

The tables given in most text-books showing the chemical composition 
of the urine cannot be regarded as absolutely indicative of the excretion as 
shown in the every-day specimens of urine. The composition of the urine 
is absolutely dependent upon the diet under normal conditions, so that a 
table to be exact must embrace the findings under a specified diet.^ Perhaps 
the most frequently quoted table is that of Parkes, which may be found in 
almost any text-book dealing with the urine. This table gives the various 
figures for the different substances excreted, but does not take into considera- 
tion the amount of the various types of foods used in the diet leading to this 
excretion. The writer, therefore, feels that it is wise to omit such a table at 
present, as we have no method of comparison of the excretion with the intake. 
The recent work of Folin upon the urine of persons both under a mixed diet 
and a nitrogen-free diet gives an "approximately complete" determination of 
the urinary constituents under absolutely fix:ed dietary conditions. The 
figures for such diets will be given under the head of each individual substance 
discussed. 

The daily urine of a healthy adult will vary between 1,200 and 1,500 c.c. 
in amount and will contain from 60 to 70 grams of total solids, of which the 
inorganic constituents form from 25 to 30 grams and the organic between 35 
and 40 grams. -"^ The inorganic constituents consist of the phosphates of 
sodium, potassium, calcium, and magnesium, the chlorids and sulphates 
of the alkali metals, various types of ammonium salts, traces of nitrates,^ cal- 

1 Centralbl. f. d. Med. Wissensch., 1876. 

^ See Long, Jour. Am. Med. Assn., 191 2, LVIII, 757; also, Long and Gephart, Jour. Am. 
Chem. Soc, 191 2, XXXIV, 1229. Kinloch, Jour. Path, and Bacteriol., 1914, XIX, 77; 
Blatherwick, Arch. Int. Med., 1914, XIV, 409. 

3 See Atkins and Wallace (Biochem. Jour., 1913, VII, 219) for a discussion of the critical 
solution point of urine. 

■* See Mitchell, Shonle, and Grindley, Jour. Biol. Chem , 1916, XXIV, 461. 



196 DIAGNOSTIC METHODS 

cium carbonate (especially under vegetable diet), and traces of iron com- 
pounds. While these inorganic substances have not hitherto been credited 
with much importance, to-day we are realizing more and more that much 
is to be learned by a careful study of the inorganic excretion.^ 

The organic substances are of especial importance both in metabolic 
work and in the diagnosis of pathologic conditions. While we do not by any 
means know everything concerning the variations in excretion of these organic 
products, yet we do know much which is helpful in our direct diagnostic work 
as well as in our study of the progress of the disease. Among the organic 
substances which are more or less normal (although not always in large 
amounts) in the urine we find the lower and higher fatty acids, oxalic acid, 
acetone, glycerophosphoric acid, a trace of glucose, lactose (especially in 
nursing mothers), carbamic acid, urea, oxaluric acid, allantoin (especially 
a few days after birth), creatinin, uric acid, purin bases, thiosulphuric acid, 
taurocarbamic acid, cystin, chondroitin-sulphuric acid, inosite, hippuric acid, 
benzoic acid, phenaceturic acid, p-oxyphenyl-acetic acid, hydro-p-cumaric 
acid, skatol-carbonic acid, conjugated sulphuric and glycuronic acids, oxy- 
proteic acid, pigments, organic iron compounds, traces of protein, and ferments. 

Under pathologic conditions we may find lactic acid, large amounts of 
acetone, aceto-acetic acid, /5-oxybutyric acid, fats, large amounts of glucose, 
levorotatory carbohydrates, r-arabinose, lecithin, cystin, putrescin, cadav- 
erin, ptomaines, oxymandelic acid, leucin, tyrosin, homogentisic acid, uro- 
leucic acid, cholesterin, cholic acid, glycocholic acid, tauro-choHc acid, various 
derivatives of phenol, hematin, hematoporphyrin, methemoglobin, other 
blood pigments, bile pigments, melanin, and protein material. 

{a) Total Solids and Total Ash. 

The estimation of the total solids of the urine is a matter of considerable 
difficulty, owing to the fact that evaporation of the urine leaves a syrupy 
residue which is dried only with much trouble and constant loss of ammonia 
formed through the action of diacid sodium phosphate upon urea in concen- 
trated solution. One may come very close to accurate results by placing a 
weighed amount of dry clean sand in a weighed platinum dish and adding 
10 to 15 c.c. of urine. This is then evaporated upon the waler-bath and 
later in the drying oven at io5°C. The dish is then placed in the desiccator 
and allowed to remain until the weight becomes constant. Knowing the 
weight of the dish and sand, the amount of the urine added, and the weight 
of the dish after the urine is evaporated the total solids may be easily calcu- 
lated. In case a large amount of residue is desired for quantitative work, 
recourse may be had to the method of Slagle,^ of adding 5 c.c. of concentrated 
H2SO4 to each liter of urine showing a specific gravity up to 1,020 and 
evaporating this to dryness. More H2SO4 must be used with the more 
concentrated urines. 

The estimation of the ash is as follows: Fifty c.c. of urine are evaporated 

^ See Baumann and Howard (Arch. Int. Med., 191 2, IX, 665) for a discussion of the 
norganic metabolism in scurvy. 

^ Jour. Biol. Chem., 1910, VIII, 77. See also, Braman, Ibid., 1914, XIX, 105. 



THE URINE 197 

to dryness over the water-bath in a weighed platinum or porcelain dish. 
The dish is then heated, while covered, over the free flame until gases cease 
to be evolved, especial care being taken not to permit sputtering of the con- 
tents. In some cases it is possible completely to incinerate the urine by long- 
continued heat. However, a more usual procedure is to treat the carbonized 
residue with distilled water, thoroughly stir the mixture and filter through 
a filter-paper whose ash is known. The contents of the dish should be washed 
onto the filter several times and the material upon the filter also washed with 
boiHng water. The filter-paper and its contents are now placed in the dish 
and completely incinerated. After this procedure the filtrate and washings 
of the original carbonized material, which contain most of the inorganic con- 
stituents, are placed in the dish and evaporated at ioo°C. to dryness, and 
then incinerated over the free flame. The dish is now placed in a desiccator 
and dried to constant weight. Knowing the weight of the dish with its con- 
tents and the original weight of the dish a simple calculation will give the 
amount of ash in the 50 c.c. of urine taken. As originally stated the inorganic 
constituents will range between 25 and 30 grams under normal conditions. 
{b) Inorganic Constituents. 

(i) Chlorids. 

The chlorids are one of th^ most important groups of organic solids 
in the urine. They are den'ved entirely from the food and, in consequence, 
the amount of excretion will depend upon the intake. The chlorids actually 
forming constituent parts of the food exist in combination with potassium and 
calcium, while those which are added as seasoning to the food are practically 
always in the form of sodium chlorid. As the amount contained in the food 
is trivial in comparison to that added, we are accustomed to regard practically 
all of the intake as sodium chlorid. This is a constituent of the serum of the 
blood and of other tissues, while the potassium salt is in more direct relation 
with the cellular elements. 

Under normal conditions from 10 to 15 grams of sodium chlorid are 
eliminated in 24 hours. The administration of a diet rich in salts will in- 
crease this amount, while a salt-poor diet will diminish the amount up to a 
certain point. If the diet be a starvation one, or an absolutely salt-free one, 
the chlorids will disappear almost entirely from the urine. The regulation 
of the metabolic activity of the system is such that a certain amount of salt 
must be retained in order to preserve the osmotic equihbrium. For this 
reason we find that withdrawal of salt from the diet does not lead to any 
appreciable diminution of the normal chlorid content either of the blood or 
tissues. An increase in the elimination of salt is practically always followed 
by a retention of salt unless a sufficient supply is furnished by the food. If 
food containing sodium chlorid be given after a period of salt-free diet, a 
portion of this salt will be retained. Conversely, we find, if the body has for 
pathologic reasons retained sodium chlorid, that an increased elimination will 
follow. This metabolic activity is intimately associated with the general pro- 
tein metabolism of the body. Any increase in the amount of circulating pro- 
tein as compared with the living protoplasm will be followed by an increased 



198 DIAGNOSTIC METHODS 

elimination of the chlorids, which have been previously retained by the living 
or active protein material of the protoplasm. This fact is shown by the rela- 
tion between the elimination of the chlorids and the total nitrogen. With 
an ordinary diet this ratio is as one to one, but in disease it may be much 
disturbed owing to chlorid retention through renal insufficiency. The blood 
and tissues of patients with nephritis show a higher chlorid content than 
those of normal individuals. We should, therefore, expect, if the law of 
increased chlorid excretion being dependent upon increased circulating 
albumin were to hold, that the chlorids would be increased along with the 
albumin in nephritis. But we find that the kidney under these circumstances 
is unable to excrete the increased amounts of salts circulating in the blood. 

A further method of withdrawing sodium chlorid from the body consists 
in the administration of large quantities of alkaline carbonates or of com- 
pounds of the alkaUes with vegetable acids. As soon as these are given the 
body becomes poor, not only in acid substances and in HCl, but also at the 
same time in sodium and potassium. Practically speaking, the body becomes 
directly impoverished in NaCl. The body may also lose chlorin when vomit- 
ing is frequent, when absorption is diminished, when the stomach is regularly 
washed out, and when diarrhea is marked. This loss may be especially noted 
in cases of hyperacidity of the gastric juice associated with vomiting. The 
chlorin in these conditions is withdrawn in the form of free acid, and in con- 
sequence the alkalinity of the tissues may be increased. 

Physiologic Variations. 

The amount of sodium chlorid excreted will depend directly upon the 
amount ingested. We may find as high as 30 grams of salt in the 24-hour 
specimen or it may be as low as 2 grams, salt-free diet reducing the elimination 
to a mere trace. The elimination may be increased by active exercise, by 
increasing the water intake and hence the water output, and by the intake 
of a large amount of vegetable food. Much more chlorid is apparently 
excreted during the day than during the night. ^ 

Pathologic Variations. 
A marked diminution of the chlorids, which may in some cases be 
almost complete, has been supposed to be pathognomonic of pneumonia. 
This, however, has been shown to be fallacious, as the same condition occurs 
in most acute febrile states, with a possible exception of intermittent fever. 
In a doubtful fever a large diminution in the amount of urinary chlorids 
might be strongly presumptive of pneumonia, but would be conclusive only 
in the presence of distinct cHnical signs of this disease. While the retention 
of these chlorids in the exudate of pneumonia may partially explain the 
diminution in the urine, it cannot explain the fact that the chlorids of the 
food are also retained. The explanation is more likely to be found, in the 
writer's opinion, in an existing renal insufficiency. ^ This same retention of 

1 See Borelli and Girardi, Deutsch. Arch. f. klin. Med., 1914, CXVI, 216; Goldberg and 
Hertz, Ibid., 201. 

2 See Snapper, Deutsch. Arch. f. klin. Med., 1913, CXI, 281; also, Hoff. Cor. — Bl. f. 
schweiz. Aerzte, 1913, XLIII, 1410. 



THE URINE 199 

chlorids will be found also in any condition in which there is a transudate or 
exudate of any considerable bulk, so that this factor must play a certain 
role. As crisis approaches in pneumonia, the chlorids of the urine will in- 
crease in favorable cases, while in those of bad prognosis no such increase will, 
as a rule, be observed. Van der Bergh believes the explanation of the dimin- 
ished urinary chlorids in pneumonia to be an attempt on the part of the blood 
to maintain its osmotic pressure, the chlorids remaining fixed in the tissues 
owing to the increase of the products of metabolism in the plasma. 

The chlorids are diminished in all acute and chronic renal diseases 
associated with albuminuria. The work of Widal upon the influence of 
chlorids upon the progress of a renal disease has brought out the facts that 
not only do we have such a chlorid retention, but that the presence of chlorids 
in the food will increase both the albuminuria and the edema of these con- 
ditions. While these facts are incontrovertible, we must take into considera- 
tion, as Richter has shown, the amount of water intake as well.^ This is 
such an important field to the cHnician that the writer would refer to other 
works giving the details of the " dechloridization'^ treatment. 

A severe diarrhea will also diminish the amount of chlorids in the urine, 
as the chlorids of the food are carried off by the bowel too quickly to permit 
of absorption. In cases of carcinoma of the stomach, in dilatation either 
from hypersecretion or stricture of the pylorus, and in some cases of ulcer 
of the stomach a diminution or even total absence of chlorids in the urine may 
be observed. 

In most chronic diseases, in anemic conditions, in rickets, and in marked 
nervous diseases, such as melancholia or mania, the amount of chlorids may 
be greatly reduced. If the output of chlorids be very low in a chronic disease, 
the prognosis becomes grave unless the diet can explain the diminution.^ 

A diminution is observed in most febrile diseases, especially in the exanthe- 
mata, while in typhoid fever the reduction is not so marked. This slight 
diminution in typhoid fever may serve as a distinguishing point in the diagno- 
sis of meningitis from typhoid fever, in the former case the diminution being 
much more marked than in the latter. In acute yellow atrophy of the liver 
the chlorids are diminished, while in cirrhosis of the liver they are somewhat 
increased.^ 

The Chlorids are increased in all conditions which have previously shown 
a retention, according to the law which has been previously discussed. We 
find thus an increase in the period of convalescence from acute febrile diseases, 
especially pneumonia. Strangely enough, the chlorids are found markedly 

1 See Fischer, Jour. Am. Med. Assn., 1915, LXIV, 325; Wilson and Hawk, Jour. Am. 
Chem. Soc, 1914, XXXVI, 137 and 1774; Leva, Ztschr. f. klin. Med., 1915, LXXXII, i; 
Lebensohn, Jour. Biol. Chem., 1915, XXIII, 513; O'Hare, Arch. Int. Med., 1916, XVII, 
711; Rackemann, Longcope and Peters, Ibid., XVIII, 496; Holt, Courtney and Fales, Am. 
Jour. Dis. Child., 1917, XIII, 73; Goto, Jour. Exper. Med., 1918, XXVII, 413; Zondek, 
Ztschr. f. klin. Med., 1919, LXXXVII, 349. 

2 See Barantschik, Deutsch, Arch, f. klin. Med., 1914, CXIV, i67;Landsberg, Ztschr. f. 
Geburtsh. u. Gynak., 1914, LXXVI, 53; Levison, Jour. Am. Med. Assn., igiSjLXIV, 326. 
For a discussion of McLean's index of chlorid excretion see section on blood. 

3 Burnham (Jour. Am. Med. Assn., 191 2, LVIII, 851) reports a case of alcoholic cirrhosis 
in which there was a constant decrease in the excretion of the urinary chlorids. 



200 DIAGNOSTIC METHODS 

increased in diabetes insipidus,^ which is associated also with the excretion 
of a large amount of urine. In epilepsy an increase may be observed follow- 
ing the attack. 

The chlorids are increased in the urine after the use of chloroform, whether 
administered internally or as an anesthetic. ^ Some of the diuretics, espe- 
cially potassium acetate, produce an increase in the urinary chlorids. 

In metabohc work it is frequently of advantage to study the effects of 
an ash-free diet upon the pathologic condition. Taylor has,^ therefore, intro- 
duced such a diet, consisting of the whites of i8 eggs, 120 grams of olive oil 
and 200 grams of crystallized sugar. The work of Goodall and Joslin^ with 
this diet confirms the earlier views that it is practically impossible to diminish 
the chlorin of the body by more than 10 to 14 per cent, and that the loss of 
water is proportionate to this. 

Estimation of the Chlorids. 

For rough clinical purposes the amount of chlorids in the urine may be 
estimated as follow^s: A few c.c. of clear, filtered urine, from which albumin 
if present is removed by heating with acetic acid, are placed in a test-tube and 
acidified with 10 drops of chemically pure nitric acid. This mixture is then 
treated with a few drops of 10 per cent, silver nitrate solution . If the chlorids 
are present in normal amount a distinct, curdy white precipitate will settle 
out. If the chlorids be increased a heavy precipitate wdll be observed, while 
if they be diminished only a cloud without any flakes will be seen. 

Quantitative Determination. 

The best method for such determination is, in the writer's opinion, the 
Arnold modification of Volhard's method.^ The principle of the test is the 
precipitation of the chlorids in a definite amount of urine by a standard 
solution of silver nitrate in the presence of an excess of free nitric acid. If 
the precipitate of silver chlorid be filtered from the solution, the excess of 
silver nitrate may be determined in the filtrate by titration with a standard 
solution of potassium sulphocyanate, using a strong solution of iron-ammo- 
nium-alum as an indicator. The urine should be as fresh as possible and 
should contain no nitrites. Albumin, unless present in very large amount, 
need not be removed. It is wise, however, in case the urine shows a high 
albumin content, to acidify the urine with acetic acid, boil, and filter off the 
precipitated albumin. In doing this one should take a definite volume of 
urine, precipitate as above, and wash the precipitate thoroughly with water 
in order to dissolve any chlorids which may have been retained by the al- 

1 See Forschbach and Weber, Ztschr. f. klin. Med., 191 1, LXXIII, 221; also, Meyer, 
Ibid., i9i2,LXXIV, 352. 

- See Graham, Jour. Exper. Med., 1915, XXII, 48. 

3 Univ. Cal. Pub., Pathology, 1904, I, 71. 

* Arch. Int. Med., 1908, I, 615. 

'" See Bayne- Jones (Arch. Int. Med., 1913, XII, 90) for a simplification of this test; also, 
McLean and SeUing, Jour. Am. Med. Assn., 1914, LXII, 1081; Blasucci, Ibid., 1399; 
Wunder, Munch. Med. Wchnschr., 1914, LXI, 2436; McLean and Van Slyke, Jour. Biol. 
Chem., 1915, XXI, 361; Seelman, Jour. Lab. and Clin. Med., 1916, I. 444; Alder, Ztschr. 
f. klin. Med., 1918, LXXXVI. 80; Halverson and Shulz, Jour. Am. Chem. Soc, 1919,^ 
XLI, 440. 



THE URINE 20 1 

bumin. The filtrate is made up to a definite volume, which represents the 
amount of urine originally taken. Thus, 20 c.c. of urine are treated as above 
and washed with sufficient water to make 50 c.c. In the test as outlined later, 
in which 10 c.c. of urine are used, 25 c.c. of this filtered albumin-free urine 
will represent 10 c.c. of original urine. ^ 

Solutions Necessary. 

(i) A solution of silver nitrate of such a strength that i c.c. is equiva- 
lent to 0.0 1 gram of NaCl or 0.00606 gram of CI. In making this solution 
29.055 grams of pure anhydrous crystallized silver nitrate are dissolved in 
I liter of distilled water. The chemically pure AgNOs as found on the 
market is perfectly reliable and needs only an accurate chemical balance for 
weighing the exact amount. It is essential that this solution should contain 
exactly the amount specified, as the accuracy of the method depends upon 
the correct strength of the volumetric solutions. 

(2) A solution of potassium sulphocyanate of such a strength that 
20 c.c. will correspond exactly to 10 c.c. of the silver solution or, in other words, 
so that 2 c.c. of the cyanate solution are necessary to precipitate the silver, 
from each c.c. of the silver nitrate solution. Other workers use solutions 
of potassium sulphocyanate of somewhat different strength, but the simplicity 
of the calculations necessary to determine the chlorids of the urine is much 
increased by such a relation between the two volumetric solutions. As po- 
tassium sulphocyanate is very hygroscopic, it is impossible accurately to 
weight the exact amount necessary to make this solution. We, therefore, 
dissolve a slight excess (9 grams) of potassium sulphocyanate in approxi- 
mately I liter of water. In order to make this solution correspond exactly to 
the silver solution it is necessary to find out how much water must be added 
to make 20 c.c. of this neutralize 10 c.c. of the silver solution. The technic 
is as follows: ten c.c. of the known solution of silver nitrate are measured 
from a buret and diluted with 50 or 60 c.c. of distilled water. Five c.c. 
of chemically pure nitric acid (specific gravity 1.2) and 5 c.c. of a strong solu- 
tion of iron-ammonium-alum are added and thoroughly mixed. This mix- 
ture is then titrated with the potassium sulphocyanate solution whose 
strength is to be determined. The principle of this titration is that the 
KCNS first combines with the AgNOs, forming a white precipitate of silver 
sulphocyanate. At the exact point at which this combination is complete 
the potassium ^sulphocyanate will combine with the iron of the indicator 
forming sulphocyanate of iron which is distinctly red in color. The titration 
is, therefore, carried to the point at which a permanent faintly reddish-brown 
color appears on shaking the mixture. The number of c.c. of the sulpho- 
cyanate solution necessary to produce this end point is then read off from 
the buret and we are ready for our correction. As the sulphocyanate solu- 
tion was intentionally made too strong, the titration should yield fewer than 
20 c.c. of this solution. Supposing 18.5 c.c. of sulphocyanate solution were 

1 If the urine be highly colored, it may be cleared with blood charcoal (see Larsson, 
Biochem. Ztschr., 1913, XLIX, 479). 



202 DIAGNOSTIC METHODS 

used we must obviously add to every 18.5 c.c. of the remaining sulpho- 

cyanate solution 1.5 c.c. of water according to the equation C = in 

which C represents the number of c.c. of water which must be added to the 
remaining solution; A' the total number of c.c. remaining after titration ;;i the 
number of c.c. consumed in one titration, and d the difference between the 
number of cubic centimeters theoretically required and that actually used 
in one titration. 

The calculation would, therefore, run as follows: 

C = '—^ '— = 79.58. We must, therefore, add to the remaining 

981.5 c.c. of pota,ssium sulphocyanate solution 79.58 c.c. of water to make the 
sulphocyanate solution of such a strength that 20 c.c. will exactly precipitate 
the silver from 10 c.c. of the AgNOs solution. 

(3). A cold saturated solution of iron-ammonium-alum. This must be 
absolutely chlorin free. 

(4) . Chemically pure nitric acid, chlorin-f ree and having practically no' 
trace of nitrous acid. Specific gravity 1.2. 

Technic. 

Ten c.c. of urine or 25 c.c. of the diluted urine from which the albumin 
has been removed are accurately measured with a pipet and placed in a 
100 c.c. volumetric flask. Five c.c. of nitric acid, 50 c.c. of water, and 20 
c.c. of the standard silver solution are then added and the mixture thoroughly 
shaken. After this mixture has stood for about 10 minutes distilled water 
is added up to the graduating mark of the flask, after which the whole is thor- 
oughly mixed and the precipitated silver chlorid allowed to settle. This 
mixture is then filtered through a perfectly dry filter into a thoroughly dry 
50 c.c. volumetric flask. This 50 c.c. of filtrate will represent, therefore, 
only 5 c.c. of urine, but the calculation made later will compensate for this. 

This 50 c.c. is then poured into a beaker of about 250 c.c. capacity and 
the volumetric flask is thoroughly washed out with water, the washings being 
added to the solution in the beaker. Five c.c. of the alum solution are then 
added and the mixture titrated with the potassium sulphocyanate solution 
to the appearance of the first permanent reddish tinge of the solution. The 
number of c.c. of sulphocyanate solution, necessary to neutralize the excess 
of silver remaining after the chlorid of silver has been filtered off, is then read 
off from the buret. 

Calculation. 
As 20 c.c. of the sulphocyanate solution are equivalent to 10 c.c. of the 
silver solution, it is evident that the number of c.c. of silver solution not used 
in the precipitation of the chlorids corresponds to the number of c.c. of sulpho- 
cyanate solution necessary to neutrahze the 50 c.c. of the filtrate. We, there- 
fore, subtract the number of c.c. of sulphocyanate solution used from 20 
(the number of c.c. of silver solution added) and obtain directly the number 
of c.c. of silver solution necessary to precipitate the chlorids in 10 c.c. of 
urine. As each c.c. of silver solution represents o.oi gram of NaCl or 0.00606 



THE URINE 203 

gram of CI, multiply these factors by the number of ex. used, the percentage 
of chlorids in the urine being obtained by multiplying the amount of chlorids 
in 10 c.c. by 10, and the total amount by simply multiplying this figure by the 
number of hundreds of c.c. in the total 24-hour specimen of urine. 

If the urine is very highly colored it is advisable to add a few drops of a con- 
centrated solution of potassium permanganate before the titration. This will 
usually decolorize the urine so that the end point will be much more distinct. 
Purdy's Centrifugal Method. 

This method, while having nothing in common with the accuracy of the 
preceding one, is very convenient and has the advantage of yielding quick 
results which are clinically available.^ 

Ten c.c. of clear, filtered, albumin-free urine are placed in a centrifuge 
tube which is graduated to 15 c.c. One c.c. of strong nitric acid and 4 c.c. 
of a 5 per cent, solution of silver nitrate are then added. The tube is shaken 
by inversion and the mixture allowed to stand for a few minutes, after which 
it is placed in the centrifuge and whirled for three minutes at the rate of 1,200 
revolutions per minute. The bulk percentage of silver chlorid is then read 
off, from which the percentage by weight both of sodium chlorid and of chlorin, 
equivalent to the precipitated silver chlorid, may be calculated. One per cent, 
by bulk represents 0.13 per cent, by weight of NaCl and 0.08 per cent, of CI. 

As previously stated, the amount of chlorin in the urine depends upon 
the amount ingested, ranging normally between 10 and 15 grams. By the use 
of Folin's standard diet, which contains 6.2 grams of CI, the excretion is found 
to be 6. 1 grams of CI in 24 hours. On the ash-free diet of Taylor the excretion 
at the end of 12 days of such a diet was 0.17 gram of CI in the 24 hours. 
(2) Phosphates. 

The phosphates occurring in the urine are the sodium, potassium, cal- 
cium, and magnesium salts of the tribasic orthophosphoric acid(H3P04). 
As previously stated, in the discussion on the reaction of the urine, normal, 
clear, acid urine contains no dibasic monacid phosphates, but all of the phos- 
phates under these conditions are of the monobasic diacid type. If the urine 
becomes less acid or amphoteric in reaction we find, however, in addition 
to the above, the disodium monohydrogen phosphate, the monocalcium phos- 
phate, and the monomagnesium phosphate; while if the urine be alkaUne 
we may find the neutral phosphates in the ascendency. It must be remem- 
bered, therefore, that the normal acidity of the urine is not strictly regulated 
by variations in the relative proportions of the monosodium dihydrogen phos- 
phate and of the disodium monohydrogen phosphate as usually stated. Be- 
sides these mineral phosphates, phosphoric acid is found in the urine in com- 
bination with glycerin as glycero-phosphoric acid, which is derived largely 
from the hydrolytic cleavage of lecithin compounds. According to Mandel 
and Oertel, Mathison,^ and Yoshimoto,^ the output of organically bound 

1 Lee Sucyoshi, Mitt, a d. med. Fak. der k. Univ., Tokyo, 1916, XIV, 425; Agasse- 
Lafont, Douris and Hayem, Bull, de I'Acad. de Med. Paris, 1916, LXXVI, 521; Achard, 
Ibid., 572. 

2 Biochem. Jour., 1909, IV, 274. 

3 Ztschr. f. physiol. Cham., 1910, LXIV, 464. 



204 DIAGNOSTIC METHODS 

phosphorus is not increased by a phosphorus-rich diet, while Kondo* 
shows that, although absolutely increased in amount, its relation to total 
P2O5 is diminished. 

The larger portion of the urinary phosphoric acid is derived from the 
food, the smaller portion coming from the metabolism of the tissue protein, 
especially the nucleins. This endogenous phosphoric acid may be of special 
importance as variations will be found depending upon the degree of destruc- 
tion of the lecithin and nuclein compounds. It is to be remembered here that 
not all of the phosphoric acid ingested is excreted, as between a fourth and 
a third of the total quantity may remain in the feces in combination with 
calcium. In studying the effects of increased ingestion of phosphates, the 
feces must, therefore, be examined quite as closely as the urine. 

Physiologic Excretion. 

The amount of phosphoric acid excreted in the 24 hours is always ex- 
pressed in terms of P2O5. The normal P2O5 excretion of the adult varies 
from I to 5 grams with an average of about 3.5 grams. The figures of Folin^ 
based upon a diet containing 5.9 grams of P2O5, show this excretion to average 
3.87 grams in 24 hours, while patients on an ash-free diet eliminate approxi- 
mately 0.75 gram. In this excretion the phosphates of sodium and potas- 
sium usually exceed those of calcium and magnesium, the former being ex- 
creted in the amounts of 2 to 4 grams in the 24 hours, the latter from i to 1.5 
grams. Little data exists regarding pathologic variations in the relation 
of these two types of phosphates so that no conclusion may at present be 
drawn. The excretion of P2O5 will vary with the food, especially with the 
amount of calcium and magnesium of the food. These bases combine in 
the intestine with the phosphoric radical forming phosphates which are 
difficultly soluble. "-^ This fact is taken advantage of by Croftan in the 
administration of calcium salts to precipitate the phosphates and thus 
diminish their activity in conditions attributable to uric acid. 

The phosphates are increased on an animal diet and diminished on a 
vegetable diet as Ziilzer has shown. During starvation an increase of the 
phosphates may be observed, as an indication of decomposition of the tissues. 
Administration of phosphates at this time will usually lead to a retention 
to counterbalance the previous loss. This same fact was observed in the 
discussion of the chlorids and may be stated as a general law, that an in- 
creased excretion is followed by a retention and a retention by an increased 
elimination. It must be stated, however, that an insufficient supply of 
phosphoric acid is not compensated for by such a great retention of phos- 
phates as of the chlorids. The organism eliminates even more phosphoric 
acid in starvation than in cases of deprivation of salt, as the decomposing 
protein sets free the salts bound up with it. Many attempts have been made 
to determine where and in what form phosphoric acid is retained in the body 
and where and from what sources the body draws upon it for excretion. It 

' Biochem. Ztschr., 1910, XXVIII, 200. See, also, Plimmer, Biochem. Jour.,1913, VII, 43,. 
2 See Wiirtz, Biochem. Ztschr., 191 2, XL VI, 103; also, Knox and Tracy, Am. Jour. Dis. 
ChUd., 1914, VII, 409. 



THE URINE 205 

is a difficult matter to determine what amount of the phosphoric acid retained 
reaches the bones, what portion is devoted to the soft tissues, and how much 
of it remains organically combined in the body (Magnus-Levy). It has been 
found that the relation between the excretion of phosphoric acid and nitrogen 
is normally about one to seven, the same relations which exist between the 
amount of nitrogen and phosphoric acid in the human muscular tissue. It 
is, therefore, plausible to assume that a retention of both nitrogen and phos- 
phoric acid will lead to a deposition of increased flesh. In starvation we find 
that this relation is markedly disturbed, the phosphoric acid being both 
relatively and absolutely increased. Such being the case the loss of P2O5 
must be largely sustained by the bones, which are relatively poor in nitrogen. 
For a full discussion of this subject the writer would refer to von Noorden's 
work on Metabolism and Practical Medicine, which gives great detail 
regarding all phases of metabolism. 

The phosphates are increased during hard muscular exercise, while mental 
exercise seems to lead to a diminished excretion of the alkaline phosphates 
and an increased output of the earthy phosphates. The ingestion of large 
quantities of water is frequently associated with an increased elimination 
of the phosphates, although this is later followed by a slight retention. 

It not infrequently happens that a freshly voided urine shows a marked 
turbidity and even precipitation due to the deposit of earthy phosphates. 
This has been supposed to be due to an increased output of the phosphates, 
but it is now known to be nothing but the natural consequence of a change 
of urinary reaction from acid to alkaline. This condition which has been 
called " phosphaturia^' would, therefore, much more appropriately be styled 
*' alkalinuria." This subject will be discussed in a later section to which 
the reader is referred.^ 

Pathologic Variations. 

A diminished elimination may be observed in cases of acute febrile 
disease, especially at the height of pneumonia. The degree of diminution 
is usually proportionate to the severity of the disease and usually lessens as 
convalescence comes on. According to Gouraud, the earthy phosphates are 
considerably reduced in pneumonia, while in tuberculous conditions the 
phosphates are increased, an interesting point in differential diagnosis. This 
retention in pneumonia as well as in the other acute febrile diseases is pos- 
sibly due to the renal insufficiency which may be very great in such conditions. 
This diminished phosphatic excretion may not always obtain in the acute 
febrile conditions, in some cases a sudden increased output being observed. 
In typhoid fever Robin believes an increased elimination during the febrile 
rise to be an unfavorable sign, while an increase during defervescence indi- 
cates a favorable prognosis. 

The phosphates appear to be diminished in most chronic diseases. In 
all renal diseases, whether acute or chronic, a diminished excretion is present 
due to the renal insufficiency. This diminished phosphoric acid excretion 

* See Dunner, Deutsch. Med. Wchnschr., 1915, XLI, 973. 



2o6 DIAGNOSTIC METHODS 

is regarded by Purdy^ as a factor almost as constant as is the excretion of al- 
bumin. ■ In gout the phosphoric acid excretion runs parallel to that of uric 
acid, decreasing immediately preceding the acute attack and rising as the 
attack subsides. In cases of pregnancy a diminished excretion is observed 
which is attributable to the withdrawal of phosphoric acid from the maternal 
organism for the purpose of the fetal bone formation. In certain bone dis- 
eases, such as osteomalacia, a diminished excretion is usually observed, 
although at times an actual increase is seen. The earthy phosphates, espe- 
cially, are diminished in these latter conditions while the alkaline phosphates 
may be increased. In cases of myositis ossificans the excretion of inorganic 
phosphates does not seem to be much affected as one might expect from the 
new bone formation. 

In cases of hystero-epilepsy the phosphates are diminished, the diminu- 
tion usually being proportionate to the intensity of the attack, while in true 
epilepsy the phosphates appear to be more or less markedly increased. It 
is in just the nervous diseases that one would expect to find much variation 
in the phosphatic excretion, but very few data are found bearing on this sub- 
ject. Folin and Shaffer find that in the periods of nervous excitement the 
relative amount of phosphoric acid is diminished, but that the absolute 
amount is little changed. 

In Addison's disease, hepatic cirrhosis, acute yellow atrophy, and chronic 
lead-poisoning we may find an extensive decrease of phosphates in the urine. 

In certain cases which show most of the symptoms of diabetes mellitus 
without any sugar output, a phosphatic increase is observed in the urine. 
This condition has been called ^^ phosphatic diabetes, ^^ and may be associated 
with the excretion of as high as lo grams" of P2O5 within 24 hours. In true 
diabetes melHtus the phosphates may be increased at one time and diminished 
at another, as there seems to be an inverse ratio between the excretion of 
sugar and that of the phosphates. 

The phosphates seem to be increased in cases of pseudoleukemia, leu- 
kemia, hemorrhagic purpura, in cases of acute or chronic inflammatory proc- 
esses of the genito-urinary tract, and in cyclic vomiting of children. 

As previously stated, the output of urinary nitrogen bears a relation of about 
seven to one to that of the phosphate excretion. This relation has been termed 
the "relative value'' of phosphoric acid and represents the amount of P2O5 cor- 
responding to 100 grams of N. Normally this ranges between 15 and 20. 

Estimation of Phosphates. 

Ten c.c. of urine are rendered alkahne with ammonia. The earthy phos- 
phates are precipitated in the form of a flocculent precipitate and may be 
roughly estimated by the volume of the precipitate. 

If this alkahnized urine be filtered and the filtrate acidified with acetic 
acid, the addition of a few drops of ferric chlorid or of uranium nitrate solu- 
tion will precipitate the alkaline phosphates. 

These methods are purely quahtative and can have no cHnical value 

^ Practical Urinalysis, Phila., 1900, p. 56. See, also, Marriott and Rowland, Arch. 
Int. Med., 1916, XVIII, 708. 



I 



THE URINE 207 

beyond giving a general idea of the relative amounts of the earthy and alka- 
line phosphates. Instead of ferric chlorid or uranium solution, magnesium 
mixture may be used for this purpose. 

Quantitative Determination. 

The best method for the estimation of the urinary phosphates is that 
of titration with uranium nitrate or acetate solution. The principle of this 
method is that phosphoric acid compounds in acetic acid solution give, on 
treatment with uranium nitrate, a yellowish-white flocculent insoluble pre- 
cipitate of uranium phosphate (U02HP04)- As a means of recognizing the 
point at which an excess of uranium solution is present in the titrated fluid, 
one may use either a solution of ferrocyanid of potassium which gives a dis- 
tinct brownish color at the end point, or, preferably, a few drops of tincture 
of cochineal, which gives a grass-green color and has the advantage that it 
can be added directly to the titrated fluid, which is not the case with the 
ferrocyanid of potassium.^ 

Necessary Solutions. 

(i) A solution of uranium nitrate or acetate of such a strength that 20 c.c. 
shall correspond to o.i gram of P2O5. It is a matter of absolute indifference 
whether the acetate or the nitrate be used, but the writer prefers the nitrate 
as this is more easily obtained in the pure state. In making up a solution 
of uranium nitrate of the above strength, one may not rely implicitly on the 
weighing, as the uranium nitrate may contain impurities or excess water 
and thus vitiate the resists. It is, therefore, necessary to have a standard 
phosphate solution against which the uranium solution may be titrated. 

The usual solution recommended by various writers is one of disodium 
monohydrogen phosphate. This salt varies in its degree of hydration and 
its solutions do not keep well. Moreover, it is absolutely necessary when this 
salt be used that a definite amount of it be taken and converted into sodium 
pyrophosphate, after which a corresponding dilution of the solution must be 
made to make it of such a titer that every 50 c.c. shall be equivalent to o.i 
gram of P2O5. In view of these facts, the writer is accustomed to follow 
the suggestion of Giles^ and use chemically pure dihydrogen monopotassium 
phosphate. This salt crystaUizes well without any water of crystalHzation 
and does not alter on exposure to the air. 

This solution is to be made such a strength that 50 c.c. corresponds to 
O.I gram of P2O5, in other words a Hter must contain 2 grams of P2O5. In 
order to find out just how much of this salt must be dissolved in a liter of 
water we must have recourse to a simple calculation. The formula of dihy- 
drogen monopotassium phosphate is KH2PO4, its molecular weight being 
136. Two molecules of this salt are necessary to yield one molecule of P2O5 
according to the equation 

2KH2PO4 = P2O5 + K2O + 2H2O. 

If, therefore, one Hter of the solution must contain 2 grams of P2O5, the 

^ See Sato, Jour. Biol. Chem., 1918, XXXV, 473, for a micro method based on this test. 
2 Sutton's Volumetric Analysis, Philadelphia, 1904, p. 294. 



208 



DIAGNOSTIC METHODS 



amount of KH2PO4 which must be dissolved in a liter is easily calculated 
from the following proportion: 



272 : 142 : : X : 2. x = 3.83 

We, therefore, dissolve 3.83 grams of dihydrogen monopotassium phosphate 
in I Hter of water and obtain directly a solution which contains 2 grams of 
P2O5 or one in which every 50 c.c. is equivalent to o.i gram of P2O5. It is 
perhaps, needless to add that this solution should be made in an accurately 
standardized volumetric flask, and at the temperature at which the flask 
is calibrated. 





Fig. 71. — Volumetric flask 



Having thus obtained our standard phosphate solution, we are now in 
a position to make up our standard uranium nitrate solution, the titer of 
which must be such that 20 c.c. corresponds to o.i gram of P2O5, or, in other 
words, one liter of which must be equivalent to 5 grams of P2O5. The 
formula of uranium nitrate is U02(N03)26H20, its molecular weight being 
502.6. Uranium nitrate combines with dihydrogen potassium phosphate 
according to the following equation: 

U02(N03)2 + KH2PO4 = UO2HPO4 + HNO3 + KNO3. 

As seen above, two molecules of the dihydrogen phosphate are necessary to 
yield one molecule of P2O5. We will, therefore, have, when uranium nitrate 
acts upon the dihydrogen phosphate, only the equivalent of }/2 molecule 
of P2O5; that is, 71 parts. As the uranium solution must contain 5 grams of 
P2O5 to the hter we may then calculate how much uranium nitrate is necessary 
to form the equivalent of such a solution by the following proportion : 



502.6 : 71 ::x : 5. 



35-39- 



THE URINE 209 

Were we absolutely certain of the purity and state of hydration of our 
uranium nitrate, all that would be necessary would be to weigh out this 
exact amount. As this is not the case, we weigh out a slight excess (35.75 
grams) and dissolve in one liter of distilled water. We are now ready to 
determine the strength of the uranium solution as follows: 

Fifty c.c. of the dihydrogen monopotassium phosphate solution are placed 
in a beaker and treated with a few drops of tincture of cochineal and 5 c.c. 
of acetic acid mixture (see below, solution 2). Some workers prefer the ad- 
dition of potassium ferrocyanid as an indicator, but this does not give as dis- 
tinct a contrast at the end point, and if tests are made by adding a drop of the 
mixture to the ferrocyanid solution on a white plate, loss of substance must 
occur. This mixture is then heated and titrated, as soon as the boiling point 
is reached, with the uranium solution until a trace of a distinct green color 
becomes permanent on stirring the mixture. Duplicate determinations are 
then made, the results of which should agree exactly with the original. The 
number of c.c. of uranium solution used is then read off and we are pre- 
pared for the calculation of the amount of water which must be added to 
standardize the solution. 

As 20 c.c. of this uranium solution should correspond exactly to 50 c.c. 
of the standard phosphate solution, we may, for the sake of example, use the 
same figures given for obtaining the dilution in the case of the sulphocyanate 
solution discussed under the heading of Chlorids. Thus if 18.5 c.c. of ura- 
nium solution were used we must add, according to the previous explanation, 
79.58 c.c. of distilled water to the remaining 981.5 c.c. of uranium solution 
in order to make every 20 c.c. equivalent to o.i gram of P2O5. 

(2) An acetic acid mixture prepared by dissolving 100 grams of sodium 
acetate and 30 grams of glacial acetic acid in sufficient water to make 1000 
c.c. This solution must be added in the determination of the urinary phos- 
phates in order to overcome the influence of the nitric acid liberated in the 
reaction and to convert any monacid phosphates into the diacid type. 

(3) An indicator, preferably tincture of cochineal prepared by digesting 
the ground cochineal bugs in 25 per cent, alcohol and filtering. This indicator 
has the advantage that it may be added directly to the solution to be titrated, 
while potassium ferrocyanid must be used by the plate method of adding 
a few drops of the solution to the indicator after each addition of uranium 
solution. 

Technic. 
Fifty c.c. of clear filtered urine are placed in an Erlenmeyer flask and 
treated with 5 c.c. of the acetic acid mixture, for the purpose of transforming 
any monacid phosphates into the diacid form and of neutralizing the nitric 
acid formed during the titration. A few drops (5 to 10) of tincture of 
cochineal are added, the mixture heated to the boiling-point, and then 
titrated as described above. It is wise invariably to run dupHcate determina- 
tions. After each addition of the uranium nitrate the precipitate is allowed 
to settle so that one may see more clearly the first trace of any green coloration 
or precipitate. 



2IO DIAGNOSTIC METHODS 

The calculation is as follows: Supposing lo c.c. of the uranium solution 
were used, the corresponding amount of P2O5 in the 50 c.c. of urine examined 
would then be found from the equation: 

20 : 0.1 : : 10 : X. x = 0.05. 

The percentage of P2O5 would, therefore, be o.i (2 X 0.05). If the total 
24-hour urine were 1,500 c.c, the total P2O5 excretion would be obviously 
1.5 grams. 

Total Phosphoric Acid. 

The above determination gives the total P2O5 excretion as far as the 
inorganic phosphates are concerned, but it does not take into consideration 
the organically bound phosphorus. This is usually very small in amount, 
but it is sometimes of value to know whether any variations exist. 

This may be determined by incinerating 50 c.c. of urine and determining 
the total phosphoric acid in the ash. This, however, is not as easily done 
as might be imagined, owing to the fact that the urinary residue is difficultly 
incinerated. The writer prefers, therefore, the method of Asboth-Folin 
which will be discussed in the section on Sulphates: The mass fused by 
this method is dissolved in hot water, the solution filtered and washed with 
hot water. This solution is then slightly acidified with HCl, treated with the 
acetic acid mixture, and titrated as in the previous determination. The 
difference between the figures obtained here and those obtained by direct 
titration of the urine will yield the amount of organically-bound phosphorus 
in terms of P2O5. The normal output of this organic phosphorus varies from 
0.0 1 to 0.1 gram in 24 hours, forming about 2 or 3 per cent, of the total P2O5. 
Phosphorus-rich diets increase the absolute amount but diminish the relative 
percentage. 

Purdy's Centrifugal Method. 

This method cannot be relied upon for accurate results in metabolic 
work, but may of be some service from the clinical standpoint. Ten c.c. 
of clear filtered urine are placed in a centrifuge tube graduated to 15 c.c. 
Two c.c. of 50 per cent, acetic acid, and 3 c.c. of 5 per cent, uranium nitrate 
solution are then added and thoroughly mixed with the urine by inversion of 
the tube. The tube is then placed in the centrifuge and operated at a speed 
of 1200 revolutions for three minutes. According to Purdy, i per cent, by 
bulk of uranium phosphate equals 0.04 gram of P2O5 in each 100 c.c. of 
urine. Each succeeding percentage by bulk increases by the figure o.oi. 
Thus a bulk percentage of five of uranium phosphate would equal 0.04 plus 
0.04, or 0.08 gram of P2O5 in each 100 c.c. These figures are much at vari- 
ance with those of Ogden, who states that "he has found that each }{q of 
a c.c. of precipitate calculated as P2O6 is equivalent to 0.0225 P^^ cent, by 
weight." Owing to these differences, the writer would suggest that the bulk 
percentage be stated as such rather than as parts by weight of P2O5. 

(3) Sulphur Compounds. 

The sulphur is present in the urine in three forms: (i) preformed or neutral 
sulphates; (2) ethereal or conjugated sulphates, sulphuric acid in combination 



THE URINE 211 

with aromatic compounds, and (3) neutral, unoxidized, or organic sulphur. 
The total output of sulphur depends essentially upon the protein metabolism, 
both of that of the tissues and of the food.^ It is to be remembered that the 
sulphur elimination is much less accurate than that of the nitrogen as an indi- 
cation of the degree of protein metabolism, owing to the fact that different 
protein substances vary in their sulphur-content. The daily excretion of 
sulphur, in terms of SO3, varies from i to 3.5 grams, when the subject is upon 
a mixed diet. Ordinarily, the ethereal sulphates form about one-tenth of the 
total output. The neutral sulphur does not vary under normal conditions 
as far as its absolute amount is concerned, but we note, on changing the diet 
to one which is relatively free in protein material, that the relative amount 
of the neutral sulphur is markedly increased. Thus, Folin finds on a diet 
containing 18.9 grams of nitrogen and 3.8 grams of SO3 the daily excretion 
of total SO3 is 3.31 grams, of which the inorganic SO3 is 2.92 (87.8 per cent, 
of total), the ethereal SO3 is 0.22 (6.8 per cent.), and the neutral SO3 0.17 
(5.1 per cent.). On a nitrogen-free diet, consisting of cream and arrowroot, 
a total SO3 excretion of 1.04 grams is noted, of which 0.63 gram (60.6 per 
cent.) is traceable to the inorganic SO3, 0.12 (11.5 per cent.) to the ethereal 
SO3, and 0.29 (27.9 per cent.) to the neutral SO3. We are, therefore, con- 
fronted with the following fact, " the distribution of the sulphur in urine among 
the three chief normal representatives, inorganic sulphates, ethereal sulphates, 
and 'neutral sulphur,' depends on the absolute amount of sulphur present." 

On the ash-free diet of Taylor we find, according to Goodall and Joslin, 
a total SO3 secretion of 0.96 gram, of which the inorganic SO3 forms 0.71 
gram (74 per cent.), the ethereal sulphates 0.05 gram (5.2 per cent.), and 
the neutral sulphur 0.2 gram (20.8 per cent.). 

It is doubtless true that practically all of the urinary sulphur is derived 
from protein metabolism, a definite relation being usually established be- 
tween the nitrogenous and sulphur output. Normally, N:S03::5:i, 
apparently regardless of whether the patient is on a nitrogen-rich or a nitro- 
gen-free diet; the absolute amounts of each, howxver, differ markedly, de- 
pending upon the diet. Folin's figures show that as the total urinary sulphur 
is reduced, the percentage represented by the inorganic sulphates sinks from 
about 90 to 60 per cent. This fact has been expressed in the above quotation 
from Fohn. 

The reduction in the inorganic sulphates must be made up by a relative 
increase in the other forms of sulphur. The ethereal sulphates have for a 
long time been held to be an accurate index of the degree of absorption of the 
products of intestinal protein decomposition. There can be no question but 
that increased intestinal decomposition is associated with increased output of 
the conjugated sulphuric acids, especially the indoxyl and skatoxyl sulphuric 
acids. These ethereal sulphates are diminished on a milk diet or on the 
cream and arrowroot diet of Folin or the vegetarian diet adopted by many of 
Chittenden's subjects. This is true especially as regards the urinary indican 
which is absolutely negative in such cases, while the total amount of ethereal 

^ See Cathcart and Green, Biochem. Jour., 1913, VII, i. 



212 DIAGNOSTIC METHODS 

sulphate is diminished only about 50 per cent. Such being the case we must 
have some other than intestinal origin for the large relative increase of the 
ethereal sulphates under a nitrogen-free diet. I quote from Folin:^ "(i) 
The urinary indican is not to any extent a product of the general protein 
metabolism, is therefore probably, as is generally supposed, a product of in- 
testinal putrefaction, and may consequently be assumed to indicate approxi- 
mately the degree of putrefaction in the intestinal tract. (2) The ethereal 
sulphates can only in part be due to intestinal putrefaction, and neither 
their absolute nor their relative amount can be accepted as an index of the 
extent to which the putrefaction is taking place in the intestines. (3) The 
ethereal sulphates, on the contrary, represent a form of sulphur metabolism 
which becomes more prominent when the food contains little or no protein." 
Here the sulphuric acid is conjugated with aromatic bodies formed from de- 
composition of tissue protein. 

Pathologic Variations. 

The sulphates, as a whole, must be increased in any condition associ- 
ated with increased protein catabolism. Thus we find in febrile conditions 
an increased output of sulphur corresponding to the intensity of the process, 
this increase being followed by a diminution as convalescence comes on. 
An increased elimination of sulphates has been observed in leukemia, diabetes 
mellitus and insipidus, progressive muscular atrophy, and following the use 
of such drugs as morphin, potassium bromid, sodium salicylate and acet- 
anilid. From the clinical standpoint the elimination of sulphates has little 
practical value, the variation in the amounts of ethereal sulphates and neu- 
tral sulphur being the chief factors of value. ^ 

While, as shown above, the ethereal sulphates are subject to great varia- 
tion, the indoxyl-potassium sulphate (indican) varying according to the de- 
gree of intestinal decomposition, we find some points of clinical interest in their 
study. As the putrefactive processes normally occur below the ileo-cecal 
valve, any condition increasing such decomposition with a consequent in- 
crease of urinary indican would indicate trouble in the lower bowel, more 
frequently of the chronic type. They are increased in cases showing abnor- 
mal intestinal absorption, as, for instance, in typhoid fever, intestinal tuber- 
culosis, peritonitis, and chronic intestinal catarrh. Obstructive jaundice is 
usually associated with increase in the output of indican as the bile seems to 
have a great influence upon putrefactive processes in the intestine. They ap- 
pear to be increased in cholera, while in ordinary diarrhea they are diminished 
in absolute amount, but may be relatively increased. In cases of gastric 
hypoacidity associated with bacterial decomposition in the stomach the urine 
may show a marked increase of indican. In acute nephritis a very intense 
indican reaction may be obtained, while in the chronic form the amount of 
indican is usually diminished. In cases of pus-formation almost anywhere 
within the system an increased elimination of ethereal sulphates may be ob- 

^ Loc. cil. 

2 See Stadtmiiller and Rcsenbloom, Arch. Int. Med., 1913, XII, 276; Ross, Ibid., 1914, 
XIII, 889; Greenwald, Ibid., 1914, XIV, 374. 



THE URINE 213 

served due to the absorption of the products of decomposition of the pus. 
This point is of some importance in differentiating purulent from non-puru- 
lent affections of various organs. The writer has seen a persistent intense 
indicanuria, associated with middle-ear infection, which cleared up completely 
after thorough drainage. 

Neutral Sulphur. 

The neutral sulphur does not have a very definite relation to the amount 
of sulphur of the intake or to the amount formed in the decomposition of 
tissue protein. While it is said to vary in amounts representing from 12 
to 15 per cent, of the total SO3 of the urine, this must be true only when the 
absolute amount of excreted SO3 is taken into consideration. By this is 
meant that although the absolute amount of neutral sulphur does not nor- 
mally vary to any great extent, its percentage relation to the total sulphur 
varies with the amount of sulphur. Thus Folin finds on an intake of 3.8 
grams of SO3 an output of 0.17 gram of neutral SO3, while on a nitrogen-free 
diet the output is 0.2. 

The nature of the neutral sulphur of the urine is somewhat uncertain, 
as we seem to have only two well-established bodies, namely, the sulpho- 
cyanates and hydrogen sulphid. The sulphocyanates are derived largely 
from the absorption of material from the saliva and represent approximately 
one-third of the total neutral sulphur. The hydrogen sulphid may be re- 
garded as a decomposition product. Besides these we find cystein, which is 
an intermediary product of normal protein metabolism, and tauro-carbamic 
acid, which is derived from the biliary material. In cases of jaundice we 
may find as high as 60 per cent, of the sulphur in the neutral form, which 
may be due to the absorption of this and other biliary material. Traces of 
chondroitin-sulphuric acid, oxyproteic acid, alloxyproteic acid, and uroferric 
acid contribute to the neutral sulphur-content of the urine. 

The greatest increase of heutral sulphur is probably associated with 
the presence of cystin which is not normally present in the urine. This is 
undoubtedly derived from abnormal protein decomposition. The sulphur 
in the neutral form may reach as high as 30 or 40 per cent, of the total, due 
to the presence of cystin. The writer will refer to a later section for a discus- 
sion of the subject of cystinuria. 

The variations in the neutral sulphur of the urine must be regarded 
as indicative of abnormal metabolic processes which are not associated with 
variations in the other types of urinary sulphur. As Folin has shown, "the 
neutral sulphur is not at all due to processes identical or similar to those which 
give rise to indican. The neutral sulphur represents products which in the 
main are independent of the total amount of sulphur eliminated or of pro- 
tein catabolized." 

Estimation of Total Sulphur (A). 

The method followed by the writer in determining the total urinary 
sulphur in terms of SO3 is the FoHn^ modification of the Asboth-Modrakow- 

1 Jour. Biol. Chem., 1906, 1, 131; Jour. Amer. Chem. Soc, 1908, XXXI, 284. See, also 
Raiziss and Dubin, Jour. Biol. Chem., 1914, XVIII, 297. 



214 DIAGNOSTIC METHODS 

sky method. Twenty-five c.c. of urine (50 c.c. if very dilute) are measured 
into a large nickel crucible of about 200 c.c. capacity and treated with 3 grams 
of sodium peroxid.^ The mixture is evaporated to a syrupy consistency and 
is then carefully heated to dryness. The latter part of this procedure requires 
about 15 minutes. The crucible is removed from the flame and allowed to 
cool. Moisten the residue with i or 2 c.c. of water, add about 7 grams of 
sodium peroxid and heat the mixture to complete fusion for about 10 minutes. 
Allow the fused mass to cool, add about 100 c.c. of water and heat to boihng 
for at least one-half hour to dissolve the alkali and decompose the peroxid. 
This mixture is transferred to an Erlenmeyer flask (about 400 c.c. capacity) 
by means of hot water and is diluted to about 250 c.c. Concentrated HCl is 
slowly added to the almost boiling solution until the oxid of nickel (formed in 
the process) just dissolves. This will require about 18 c.c. of acid for 8 grams 
of Na202. After boiling for a few minutes, the solution should be perfectly 
clear. If it is not clear too much water or too little peroxid were used in the 
last fusion. Allow the mixture to cool and filter from the insoluble residue, 
if there is any. To this clear acid solution add 5 c.c. of very dilute (i to 4) 
alcohol and boil for a few minutes to remove the last traces of chlorine, which 
are formed on acidifying the solution. Now add, drop by drop, 10 c.c. of a 
10 per cent, barium chlorid solution and allow the precipitate of BaS04 to 
settle out for two days in the cold. At the end of this time the precipitate 
is filtered off, using either ash-free filters, or preferably, weighed porcelain 
Gooch crucibles with properly prepared asbestos mats. Wash the precipitate 
with about 250 c.c. of cold water, dry and ignite for 15 minutes. In this heat- 
ing of the Gooch crucible, the flame must touch neither its perforated bottom 
nor its sides. If the crucible be placed upright upon the cover of a platinum 
crucible, which rests upon a platinum triangle the flame may be applied 
directly to the bottom of the platinum. After the heating, the crucible is 
placed in the desiccator, is allowed to cool and is weighed. Knowing the 
weight of the crucible and mat before and after this process, a simple 
calculation yields the amount of BaS04 obtained from the urine taken. 
If the filter-paper be used, the precipitate is washed as previously mentioned. 
Transfer the filter-paper and precipitate directly to a weighed platinum or 
porcelain crucible, add 3 or 4 c.c. of alcohol and ignite, to dry and partially 
burn the filter-paper. Heat the residue until complete incineration occurs 
and the ash becomes colorless. Cool the crucible in a desiccator and 
weigh. If the amount of BaS04 obtained by either of these methods be 
multiplied by 0.3429, the result is the amount of SO3 in the urine taken. 
A simple calculation yields the percentage and total amount excreted. 
Determination of Total Sulphates (B) . 

Folin's Methods. 
Under this head we determine the neutral (preformed or inorganic) and ethe- 
real sulphates. Folin^ has advanced two methods, the first of which is preferable. 

^ Benedict, Denis, and Schmidt use, as the oxidizing agent, a solution of copper nitrate 
and potassium chlorate. See Tsuji, Biochem. Jour., 1915, IX, 439; Drummond, Ibid. 
492; Givens, Jour. Biol. Chem., 1917, XXIX, 15. 

^Loc. cit. See, also, Rosenheim, Biochem. Jour., 1914, VIII, 143. 



I 



THE URINE 215 

1. Precipitation in the Cold. — 25 c.c. of urine and 20 c.c. of dilute hydro- 
chloric acid (i to 4), or 50 c.c. of urine and 4 c.c. of concentrated HCl, are 
gently boiled in an Erlenmeyer flask (capacity about 200 c.c.) for 20 to 30 
minutes (never less than 20). To prevent loss of material it is wise to keep 
the flask covered with a small watch-glass during the boiling. The flask 
is cooled for two or three minutes in running water and the contents are then 
diluted with cold water to about 150 c.c. To this solution are then added 
10 c.c. of a 5 per cent, barium chlorid solution, care being taken not to shake 
or stir the contents during the addition. At the end of an hour or more, the 
mixture is shaken up and filtered. The rest of the determination is as out- 
lined above. 

2. Precipitation in the Heat. — The boiling of the urine with hydrochloric 
acid is conducted exactly as in the preceding method. At the end of 20 to 
30 minutes, the boiling urine is diluted to about 150 c.c. with hot water. The 
mixture is heated once more to the boiling point, is removed from the flame, 
and immediately precipitated by the addition of 5 c.c. of 10 per cent, 
barium chlorid solution, which must be added drop by drop. The mixture 
is allowed to stand for 2 hours in order to cool. The remainder of the process 
is as previously given. 

Determination of Inorganic (Neutral) Sulphates (C). 

About 100 c.c. of water (not less), 10 c.c. of dilute hydrochloric acid 
(i to 4), and 25 c.c. of urine (50 c.c. if dilute, when a correspondingly smaller 
amount of water is taken) are measured into an Erlenmeyer flask of about 200 
c.c. capacity. 10 c.c. of a 5 per cent, solution of barium chlorid are added, 
using the drop method, the urine solution not being shaken or disturbed dur- 
ing this addition. At the end of an hour or more, the mixture is shaken and 
filtered. The remaining steps are given above. The figure obtained here 
represents the amount of SO3 referable to the preformed sulphates. 

The ethereal sulphates (D) need not be determined separately as the 
difference between the total sulphates (B) and the inorganic sulphates (C) 
will represent the amount of these ethereal sulphates in the specimen ex- 
amined. If the SO3 referable to the total sulphates (B) be subtracted from 
the SO3 of the total sulphur (A) the remainder represents the neutral sulphur 
in terms of SO3. 

Purdy's Centrifugal Method. 

Ten c.c. of clear urine are placed in a centrifuge tube and to it are added 
5 c.c. of barium chlorid mixture, consisting of four parts of barium chlorid, 
one part of concentrated hydrochloric acid, and 16 parts of distilled water. 
The tube is inverted to insure mixing of the reagent and urine and is allowed 
to stand for a few minutes. It is then placed in a centrifuge and whirled 
three minutes at the rate of 1,200 revolutions per minute. Each per- 
centage of BaS04 by bulk represents approximately 0.25 per cent, of SO3 by 
weight. This result can, of course, not be as accurate as the preceding, but 
has some clinical advantage. 



2l6 DIAGNOSTIC METHODS 

(4) Carbonates. 

A freshly voided specimen of urine may contain small quantities of car- 
bonates and bicarbonates and some free carbonic acid.^ The amount of free 
carbonic acid varies with the degree of acidity of the urine and the amount of 
carbonate-forming material in the food. It has been found that vegetable 
foods are almost always productive of an alkaline urine, owing to the fact 
that the organic acids of the vegetables may be converted into carbonates 
and excreted as such. The carbonate which most frequently forms as a sedi- 
ment in the urine is calcium carbonate which will be treated in a later section. 

If the urine be acidified and a stream of air passed through this acidified 
urine into a vessel containing a solution of barium hydrate, the carbon-dioxid 
liberated by the acids will unite with the barium forming barium carbonate 
which may be filtered, dried, and weighed. 

(5) Sodium and Potassium. 

These metals exist in the urine in the form of the oxids Na20 and K2O, 
the former being present in amounts of 4 to 7.5 grams, the latter varying 
from 2 to 4 grams. The normal relation between the excretion of these 
products is as 5 to 3. 

The sodium is largely derived from the addition of sodium chlorid to 
the diet, while the potassium is a constituent of most vegetable foods. We 
find, therefore, that both of these substances depend largely upon the diet 
in normal cases, while in pathologic conditions the potassium may be excreted 
in larger amounts.^ Thus we find in fever the potassium salts predominate 
over the sodium compounds up to the time of crisis, after which the sodium 
salts again assume their normal proportions. Increased exercise as well as 
increased decomposition of protein from pathologic causes will tend to increase 
the potassium of the urine. From the clinical standpoint the quantitative 
determination of the sodium and potassium of the urine has little significance. 

(6) Calcium and Magnesiimi. 

Both of these alkali-earth metals are excreted in the urine largely in 
the form of the phosphates. Calculated as the oxids, the calcium excretion 
(CaO) varies from o.i to 0.3 gram and the magnesium (MgO) from 0.15 
to 0.4 gram in 24 hours, the relation between the calcium and magnesium 
excretion being about i to 1.5, although Nelson and Burns have shown that 
any given individual may exhibit a constant behavior as to whether calcium 
or magnesium shall be greater in the urine voided. 

The chief source of these compounds is the food, but it must be remem- 
bered that only a small portion of these substances is excreted in the urine. 
Calcium and, to a less extent, magnesium form compounds with phosphoric 
acid in the bowel and are excreted without being absorbed. Even if these 
compounds be injected subcutaneously the excretion is largely into the intes- 
tine. We should, therefore, study much more closely the calcium metabolism 
by examination of both the feces and urine. 

1 Denis and Minot, Jour. Biol. Chem., 191 8, XXXIV, 569, report the total CO2 output 
in 24 hours as varying from 20 to 211 c.c. 

2 See Blumenfeldt, Inaug. Dissert., Berlin, 191 2; also, Ztschr. f. exp. Path. u. Therap. 
1913, XII, 523; Hamburger, Biochem. Ztschr., 1915, LXXI, 415. 



THE URINE 217 

Little is known regarding the output of either of these metals, but what 
work has been done is more closely connected with the calcium excretion 
than that of magnesium. During starvation calcium oxid is increased both 
relatively and absolutely, its probable source being the bones. It is increased 
to some extent by exercise and is diminished after the administration of alka- 
lies. In chronic diseases we note an increase due, probably, to inanition. In 
tubercular conditions the calcium output is occasionally found to be dimin- 
ished, while some cases of a marked increase have been noted. In diabetes 
mellitus, as in other conditions associated with an acidosis, the output of 
CaO may be greatly increased, thus running parallel to the ammonia output. 
In a case of myositis ossificans studied by the writer there seemed to be no 
great variation in the calcium output, while the magnesium content of the 
urine was markedly reduced. Austin has reported similar findings. Mc- 
Crudden and Fales report (Arch. Int. Med., 191 2, IX, 273) a loss of calcium 
and a retention of magnesium in non-puerperal osteomalacia. The same 
authors (Jour. Exper. Med., 191 2, XV, 450) find the calcium excretion in the 
urine, in cases of intestinal infantilism, to be almost negligible. Peabody 
(Jour. Exper. Med., 1913, XVII, 71) shows a retention of calcium and a 
normal excretion of magnesium in pneumonia.^ 

Quantitative Determination. 
Calcium. 

McCrudden^ has recently introduced a method for this determination 
which, in my opinion, yields the best results. If the urine be alkaline it must 
be made neutral or slightly acid with HCl and be filtered. If turbid or 
faintly acid to litmus, add 10 drops of concentrated HCl. Two hundred c.c. 
of the clear acid urine are treated with 10 c.c. of a 2.5 per cent, oxalic acid 
solution and 8 c.c. of 20 per cent, sodium acetate solution. Allow the mixture 
to stand over night at room temperature or shake vigorously for 10 minutes. 
Filter o£E the precipitate of calcium oxalate and wash it Cl-free with 0.5 
per cent, ammonium oxalate solution. Save the filtrate for the determination 
of magnesium. The precipitate of calcium oxalate is then dried on the filter- 
paper at ioo°C. and is placed in a weighed platinum crucible and burned over 
the Bunsen flame until the filter-paper is completely ashed. The blast-lamp is 
then applied and the crucible and contents blasted for 15 or 20 minutes. The 
crucible is dried in the desiccator and weighed, the increase in weight repre- 

^ See, also, Voorhoeve, Deutsch. Arch. f. Min. Med., 1913, CXI, 29. For various studies 
of the metabolism of calcium see Diller and Rosenbloom, Am. Jour. Med. Sc, 1914, 
CXLVIII, 65; Bergeim, Stewart and Hawk, Jour. Exper. Med., 1914, XX, 218 and 225; 
Bookman, Am. Jour. Dis. Child., 1914, VII, 436; Rosenbloom and Cohn, Arch. Int. Med., 
1914, XIV, 126^; Kleinschmidt, Berl. klin. Wchnschr., 1915, LII, 29; Da Costa, Funk, 
Bergeim and Hawk, Pub. from Jefferson Med. Coll. and Hosp., 1915, VI, i; Bookman and 
Epstein, Am. Jour. Med. Sc, 1916, CLI, 267; Blatherwick, Ibid., 432; Rosenbloom, Ibid., 
CLII, 256; Vance, Ibid., 693; Kahn and Kahn, Arch. Int. Med., 1916, XVIII, 212; 
Nelson and Williams, Jour. Biol. Chem., 1916, XXVIII, 231; Nelson and Burns, Ibid., 
237; Givens and Mendel, Ibid., 1917, XXXI, 421, 435 and 441; Stehle, Ibid., 461; Sawyer, 
Baumann and Stevens, Ibid., 1918, XXXIII, 103; Givens, Ibid., XXXIV, 119; Sherman, 
Gillett and Pope, Ibid., 373; Givens, Ibid., XXXV, 241; Goto, Ibid., XXXVI, 355; 
Krieble and Bergeim, Ibid., 1919, XXXVII, 179. 

2 Jour. Biol. Chem., 1910, VII, 83; Ibid., 1911, X, 187. See, also, Lyman, Ibid., 1915, 
XXI, 551; Mayer, Presse med., 191 7, XXV, 61. 



2l8 DIAGNOSTIC METHODS 

senting the calcium oxid (CaO) in the 200 ex. of urine. A simple calculation 
will yield the amount of CaO in the 24-hour specimen. 
Magnesium. 

The filtrate and washings obtained in the above determination are treated 
with one- third the volume of 25 per cent, ammonium hydrate. A precipitate 
of magnesium ammonium phosphate (NH4MgP04) occurs, which is allowed 
to settle for several hours, is collected on an ash-free filter, is thoroughly 
washed with water containing one-third its volume of ammonia, and is dried 
in the oven at ioo°C. The filter-paper and precipitate are placed in a 
weighed platinum crucible and burned until the filter-paper is completely 
destroyed, after which the crucible and contents are heated over the blast- 
lamp for 15 minutes. This heating converts the magnesium ammonium 
phosphate (NH4MgP04) into Magnesium pyrophosphate fMg2P207); one 
part of magnesium pyrophosphate represents 0.36243 part of MgO. All that 
is necessary, therefore, is to multiply the amount of magnesium pyrophos- 
phate by the above factor to obtain the amount of MgO in 200 c.c. of urine. 

(7) Iron. 

Iron is practically always present in the urine in organic combination. 
The amount actually present is very small, being given by Magnier as vary- 
ing between 3 and 11 mg. to the Hter, while Neumann and Mayer find the 
normal output to average 0.983 mg. The urinary iron has Uttle clinical 
significance. It is increased in fever, in malaria, diabetes mellitus, and 
pernicious anemia, the amounts in some cases running as high as 20 mg. 
during the 24 hours. 

Any method for the estimation of the urinary iron must be very dehcate 
and very accurate. The urine must be completely incinerated, the method 
of Neumann^ employing a mixture of concentrated sulphuric and nitric acid 
being by far the best.^ 
(c) Organic Constituents, 
(i)- Nitrogenous Bodies. 
(a) Total Nitrogen. 

From the metaboHc standpoint the estimation of the total nitrogen of 
the urine is one of the most important features of its chemical examination. 
The excretion of total nitrogen varies with the amount of nitrogen of the 
food and with the degree of tissue metabolism. Normally, the system so 
adapts itself to the nitrogenous intake that the excretion of nitrogen, in the 
urine, feces, perspiration, etc., is equal to the intake. In other words, a 
normal person is in nitrogenous equilibrium. This subject of nitrogenous 
equilibrium is of such great importance and the factors which influence it so 
varied that the writer feels that it will be unwise to discuss it briefly for fear 
of not presenting it clearly to the student's mind. As a fiill discussion of this 
subject would be too extensive for the scope of this work the writer must be 
content with reference to the admirable discussion by Magnus-Levy^ in von 
Noorden's Hand-book of Pathology of MetaboHsm. 

^ Arch. f. Anat. u. Physiol., Physiol. Abth., 1900, S. 159. 

2 See Goodman (Jour. Biol. Chem., 191 2, XII, 37) for the urinary iron in pneumonia. 

2 Chicago, 1908. 



THE URINE 



219 



The total nitrogen of the urine may be taken as a direct index of the pro- 
tein metabolism. Upon a starvation diet, or one from which the nitrogenous 
factors have been eliminated, we find a gradual reduction in the amount of 
urinary nitrogen. From about the fourth day of starvation the excretion 
becomes practically constant and continues until severe tissue-decomposition, 
as a sign of impending death, occurs. If, at this time, a diet rich in nitrogen 
be given, a certain amount of the intake will be retained, but not all. If this 
increased nitrogenous diet be continued a certain portion will be retained 
each day until the system again assumes a condition of nitrogenous equilib- 
rium, the output equaling the intake. It is to be remembered in this connec- 
tion that the carbohydrates and fats of the diet both have a certain direct 
influence in diminishing the protein metabolism. 

The normal amount of total nitrogen of the urine, with the subject upon 
a mixed diet, varies between 10 and 16 grams per day. The work of Chit- 
tenden^ has proven that this is much too high for individuals who desire to 
get the most out of their system with the least possible work. In other words 
"physiological economy" is much better subserved by a diet yielding from 5 
to 6 grams of total nitrogen. The subjects of his experiments showed normal 
activities, both mental and physical, and at the end of the experiments felt 
much better than before them and had gained in weight. Folin^ in his work 
has shown that the normal excretion of six subjects, each observed for a period 
of five days, was 16 grams of total nitrogen on a diet of 119 grams of protein 
yielding 18.9 grams of nitrogen. On a nitrogen-free diet the excretion aver- 
aged 3.6 grams. He says: "It may, therefore, be positively stated, as a 
principle in the chemistry of metabolism, that the distribution of the nitrogen 
in urine among urea and the other nitrogenous constituents depends on the 
absolute amount of total nitrogen present." 

The subject of the distribution of the various nitrogenous products of the 
urine has been much changed by this work of Folin and of Chittenden. 
This distribution or, as it is better called, the "nitrogen partition of the urine," 
varies according to the diet. The following table shows the excretion of[the 
various nitrogenous constituents and their percentage relations to the total 
nitrogen under a mixed diet and under one which is nitrogen free. 



Excretion in grams. 



Percentage of total N. 





Mixed 


N-Free 


Mixed 


N-Free 




Diet. 


Diet. 


Diet. 


Diet. 


Nitrogen, 


16.00 


3.60 


100.00 


100. 


Urea^^N, 


13-90 


2.20 


86.87 


61.7 


Ammonia N, 


0.70 


0.42 


4.37 


II-3 


Creatinin N, 


0.58 


0.60 


3.63 


17.2 


Uric Acid N, 


0.12 


0.09 


0.75 


2.5 


Undetermined N, 


0.70 


0.29 


4-37 


7.3 



1 Physiological Economy in Nutrition, New York, 1905; Mendel, Jour. Am. Med. Assn., 

1914, LXIII, 819; Benedict, Jour. Biol. Chem., 1915, XX, 263; Pepper and Austin, Ibid., 

1915, XXII, 81; Osborne and Mendel, Ibid., 241; Johnston and Veeder, Am. Jour. Dis. 
Child., 1917, XIII, 404; Richet, C. R. soc. de biol. de Paris, 1918, LXXXI, 133. 

2 Loc. cit. 



2 20 DIAGNOSTIC METHODS 

It will thus be seen that the total nitrogen excretion of the urine is made 
up of several factors. The principal points to be gained from the above table 
are that the urea, on a nitrogen-free diet, is markedly reduced. This reduc- 
tion must naturally be made up by increase in the other factors. We find 
the ammonia and especially creatinin markedly increased from the percentage 
standpoint, although both are absolutely diminished. We have, therefore, a 
distinctly endogenous nitrogen metabolism as well as an exogenous one. The 
urea content should, consequently, be considered as of direct importance in 
estimating the degree of protein tissue metabolism, although not as usually 
taught; This urea output cannot at the present time be considered as 
representing from 85 to 90 per cent, of the total endogenous nitrogen, but 
should be regarded more properly as representing between 60 and 65 per cent. 
It is true that the urea as found under mixed diets gives this higher figure, 
but at least 20 per cent, of this must be placed against useless activity on the 
part of the system. In other words, the intake of sufficient nitrogen to yield 
a urea excretion amounting to 85 per cent, of the total N must be considered 
unnecessary. This is a somewhat enlarged expression of the fact that most 
people eat much more than is utilized by the system.^ 

It is seen, therefore, that in the general run of urine examinations the urea 
output does not represent to us the extent of the tissue metabolism, as or- 
dinarily we have not sufficiently controlled the diet. The above table pre- 
sents a remarkable percentage increase in the amount of creatinin. The 
figures of Folin show that the absolute quantity of creatinin eliminated, 
whether upon a nitrogen-rich or on a nitrogen-free diet, is remarkably con- 
stant for the same individual. Although this is influenced to a slight extent 
by the diet, it is so slight that it can be disregarded (see Creatinin). 
Physiologic Variations. 

An increase in the total nitrogen is observed after a heavy protein meal. 
As previously stated, in starvation the nitrogen of the urine becomes constant 
after about the fourth day. This nitrogen excretion observed in starvation 
is, however, less than the minimum amount that must be given in the form of 
protein in order to maintain a nitrogenous equilibrium. As a rule, the 
amount of protein taken in is much in excess of the requirement of the sys- 
tem, so that the amount excreted probably represents protein which has never 
become a part of the system. For this reason the fallacy of considering urea 
as a direct representative of the protein metabolism of the tissues becomes 
evident. The intake of protein is, in reality, readjusted to suit the actual 
needs of the body, so that the urea can represent only a portion, about 60 per 
cent., of the total metabolic activity of the tissue protein. A physiological 
increase in the excretion of nitrogen is observed in the infant for four or five 
days after birth. The nitrogen excretion is increased when the intake of 
water has been greater than normal. This fact should be borne in mind in 
metabolic experiments in which the intake of water should be quite as much 
regulated and as well known as the intake of other substances. 

Physiologically, a diminished output of nitrogen is observed on a low 
^ See Mendel and Lewis, Jour. Biol. Chem., 1913, XVI, 19, 2>7 and 55. 



THE URINE 221 

nitrogen diet and also on a diet rich in carbohydrates and fat, as these latter 
substances provide the greater part of the necessary energy. The system 
must then utiHze its own protein. Increased exercise is supposed to give 
a slight increase in the nitrogen output owing to increased muscular activity, 
but it is to be said that the loss of water through the increased perspiration 
may be such a factor in diminishing the urinary nitrogen that no increase 
may be observed. Certain medicaments as quinin and opium will usually 
diminish the output of nitrogen. 

Pathologic Variations. 
Increased Excretion. 

Perhaps the most marked increase in the urinary nitrogen is observed in 
the acute febrile infections.^ This increase is not due to the temperature per 
se nor is there any parallelism between the urinary nitrogen and the degree 
of temperature. Whether or not this increase in nitrogen can be directly 
traced to the effects of the toxins produced by the organisms causing the dis- 
ease is still unsettled. That this cannot be the only element is proven by the 
fact that Krehl and Matthes have shown that more protein is destroyed in 
the so-called aseptic fever than is the case in the normal organism under simi- 
lar conditions, dietetic and physical. In some febrile conditions we find that 
the elimination of nitrogen may be reduced during the febrile period, while 
about the time of crisis a very marked output of urinary nitrogen may be ob- 
served. This is the well-known epicritical elimination of nitrogen. In the 
fever associated with acute nephritis the urinary nitrogen is not increased, but 
is rather diminished owing to the renal insufficiency as well as to the edema 
which occurs. A toxogenic decomposition of protein is found in cases of car- 
cinoma, pernicious anemia, chronic tuberculosis, leukemia, scurvy, and es- 
pecially in exophthalmic goiter. In cases of acute yellow atrophy and phos- 
phorous poisoning the total nitrogen may be increased, but the percentage of 
urea will be very much diminished. 

In cases of diabetes mellitus the nitrogen excretion is usually much in- 
creased, due more to the effect of the increased nitrogenous diet than to 
increased endogenous protein metabolism. Likewise, in diabetes insipidus 
a large increase in urinary nitrogen may be observed. An increase is oc- 
casionally observed in cases of nephritis owing to the large albumin content 
of the urine. During the progress of absorption of an exudate a very high 
excretion may be observed, the resolution of a pneumonic exudate, for in- 
stance, being easily followed by the variations in the urinary nitrogen. ^ 

Diminished Excretion. 

A diminution in the nitrogen excretion is usually observed in convales- 
cence from acute and chronic conditions. This is probably due to the at- 
tempt on the part of the system to make up for the losses incurred during the 
active progress of the disease. It may be diminished in conditions in which 
the absorptive power of the intestine is much reduced. If the oxidative 

^ See Sharpe and Simon, Jour. Exper. Med., 1914, XX, 282. 

2 See Matthews and Miller, Jour. Biol. Chem., 1913, XV, 87; also, Gammeltoft, Habilita- 
tionschr., Kopenhagen, 19 13. 



222 DIAGNOSTIC METHODS 

powers of the system are very much reduced as the result of chronic conditions, 
the urinary nitrogen will usually be much diminished. In cases of nephritis, 
both acute and chronic, a large diminution in the urinary nitrogen is observed. 
This is due primarily to the renal insufficiency and to the associated dropsy. 
In such conditions a marked increase may be observed in the fecal nitrogen, 
especially when marked diarrhea is a complicating factor. These periods of 
retention of nitrogen in nephritis may alternate with periods of increased 
elimination, so that examinations at different periods may show greatly con- 
flicting results. When the water output of the urine is largely diminished, 
as a result of transudation, of exudation, or of increased perspiration, the 
total nitrogen may be reduced.^ 

Estimation of Total Nitrogen (Kjeldahl). 

The principle of this method is as follows i^ The nitrogenous constituents 
of the urine are oxidized by various oxidizing agents into ammonia. This 
ammonia is converted into ammonium sulphate by the sulphuric acid which 
is added at the same time as the oxidizing agents. After the preliminary de- 
composition and oxidation of the organic nitrogen into ammonium sulphate, 
free ammonia may be liberated by the action of strong sodium hydrate and 
distilled into a standard acid solution. Knowing the strength of the acid 
solution, one may then titrate the remaining acid with a standard alkali solu- 
tion and determine how much ammonia has combined with the acid. One 
c.c. of tenth-normal sulphuric acid, used by the ammonia liberated in the 
distillation, represents 0.001401 gram of nitrogen. 
Technic. 

Five c.c. of urine are accurately measured, either with a pipet or buret, into 
a Kjeldahl flask of Jena glass of 800 c.c. capacity. Ten c.c. of concentrated 
sulphuric acid and approximately i gram of copper sulphate are added, the 
flask placed in a hood and heated over a low flame until white fumes of sul- 
phuric acid are given off (Gunning's modification). Five grams of potassium 
sulphate^ are then added and the mixture heated with an increased flame to 
boiling for one-half to three-fourths of an hour. The solution should have 
lost every trace of a yellowish color and should have become by this time a 
clear bluish-green. The worker should be cautioned regarding the fumes 
given off in this process and conduct his work only in a hood with a good draft. 
It is frequently necessary to wash down the carbon from the sides of the vessel 
by shaking the fluid in such a way that the carbonized material is carried down 
to the bottom of the flask. One should be cautious lest he lose some of the 
liquid in this manipulation, which would not only throw out his determination 

^Minot, Bull. Johns Hopkins Hosp., 1914, XXV, 332; Hefter and Siebeck, Deutsch- 
Arch. f. klin. Med., 1914, CXIV, 497; Frothingham and Smillie, Arch. Int. Med., 1915, XV, 
204; Murray, Brit. Med. Jour., 1915, I, 151; Abderhalden, Ztschr. f. physiol. Chem., 1915, 
XCVI, i; Mosenthal and Richards, Arch. Int. Med., 1916, XVII, 329; Wilson, Bull. Johns 
Hopkins Hosp., 1916, XXVII, 121; Foster and Davis, Am. Jour. Med. Sc, 1916, CLI, 
49; Losee and Van Slyke, Ibid., 1917, CLIII, 94; Jeans, Am. Jour. Dis. Child., 1917, XIII, 
145; Schamberg and Raiziss, Jour. Cut. Dis., 191 7, XXXV, 135; Lewis, Jour. Biol. Chem., 
1917, XXXI, 363. 

2 See Folin and Farmer (Jour. Biol. Chem., 191 2, XI, 493) for a micro-chemical method 
based on this Kjeldah! process; Gradwohl and Blaivas* Jour. A. M. A., 1916, LXVII, 809. 

'•^ See Scott and Meyers, Jour. Am. Chem. Soc, 1917, XXXIX, 1044. 



THE URINE 



223 



but might result in a very severe burn should any of the material fall upon him. 
The mixture is allowed to cool completely before the further steps of the 
determination can be taken. Most workers advise at this juncture the trans- 
ference of the material from the first flask into a second distilling flask. The 
writer has convinced himself that this procedure is not only unnecessary but 
is even unwise, as the transference may result in slight loss of material. He 
is, therefore, accustomed to use the same flask both for the oxidation and 
distillation. 

After the mixture has cooled the neck of the flask is thoroughly washed 
with a stream of distilled water so that every trace of material may be carried 
from the neck into the body of the flask. Sufficient additional water is added 
to bring the total up to approximately 250 c.c. A little talcum powder, a few 
pieces of pumice stone, or a few pieces of granulated zinc may then be added 




Fig. 72. — Kjeldahl's nitrogen apparatus. 

to prevent bumping of the contents when sodium sulphate separates out later 
in the process. Fifty c.c. of 40 per cent, sodium hydrate are then added for 
every 10 c.c. of sulphuric acid used in the original oxidation. Care should be 
taken in adding this strong alkali that none of it touches the upper portion of 
the neck of the flask. The alkalinized mixture is then shaken and connected 
with a Fresenius bulb which is attached to a Liebig condenser as shown in the 
accompanying cut. The outlet tube passes into an Erlenmeyer flask which 
should contain 50 c.c. of tenth-normal sulphuric acid. In cases with abnor- 
mally high nitrogen values it may be necessary to use a larger quantity than 
50 c.c. of standard acid, but the writer has found only two instances in over 
1,500 determinations in which an increased amount was necessary. The con- 
nection of the distilling flask to the bulb and condenser should be done rapidly 
to avoid any possible loss of ammonia. 



2 24 DIAGNOSTIC METHODS 

The distilling flask is heated slowly at first and the heat increased only 
after boiling has become regular. If heated too quickly, spurting of the 
liquid may occur and traces of alkali be carried into the bulb and thence over 
into the standard acid. The distillation should continue until about 150 c.c. 
have been distilled over, which will take from 20 to 30 minutes. 

The writer has observed that bumping of the mixture rarely occurs, under 
the conditions outlined above, before the ammonia is completely driven over. 
This can, however, not be taken as an absolute sign that every trace of ammo- 
nia has been distilled off. In order to see whether such is the case, one must 
test the outlet tube with a piece of moist red litmus-paper which will turn blue 
in the presence of traces of ammonia. If all ammonia has not been given off, 
the distillation must be continued until such is the case. If no more ammonia 
is being evolved, the distilling flask is disconnected from the bulb so that no 
suction may draw the standard acid into the condenser. The connecting bulb 
is removed and the tube of the condenser washed with a spray of distiUed 
water so that any material adhering may be washed into the standard acid. 
The outlet tube is disconnected and washed both internally and externally 
into the standard acid solution.^ 

The standard acid solution is then titrated with tenth-normal sodium hy- 
drate solution, using cochineal, methyl-orange, alizarin-red, or methyl-red as 
an indicator. The writer prefers the use of the latter. As each c.c. of tenth- 
normal alkaU is equivalent to each c.c. of tenth-normal acid, we subtract, from 
the original number of c.c. of acid (50), the number of c.c. of standard alkali 
used to neutralize the remaining standard acid. The difference gives us the 
number of c.c. of standard acid neutralized by the ammonia given off in the 
distillation. As each c.c. of tenth-normal acid is equivalent to 0.001401 gram 
of nitrogen, we multiply this factor by the number of c.c. of acid neutralized 
by the ammonia and obtain the amount of total nitrogen in 5 c.c. of urine. 
This result multiplied by 20 yields, of course, the percentage of total nitrogen, 
which may be changed into the actual total amount of nitrogen by multiply- 
ing it by the number of hundreds of c.c. in the 24-hour specimen. It goes 
without saying, that the reagents used in this determination must be 
ammonia-free or, at least, that their ammonia content be known. 

Folin and Wright's Simplified Kjeldahl Method. 
This method^ has the advantages that it requires very little equipment 
and that the determination can be finished easily in 20 to 25 minutes. The 
hydrolyzing-oxidizing reagent used consists of the following mixture: 50 
c.c. of 5 to 6 per cent, copper sulphate solution, 300 c.c. of ^'syrupy" phos- 
phoric acid (about 85 per cent, strength), and 100 c.c. of concentrated 
sulphuric acid. 

Technic. 

Transfer 5 c.c. of undiluted urine to a 300 c.c. Kjeldahl flask, preferably 

a Pyrex flask. Add 5 c.c. of the above digestion mixture, 2 c.c. of 10 per 

cent, ferric chlorid solution, and 4 to 6 small pebbles or pieces of granite to 

1 See Dakin and Dudley, Jour. Biol. Chem., 1914, XVII, 275 
-Jour. Bio!. Chem , 1919, XXXVIII, 461. 



THE URINE 225 

prevent bumping. Boil this mixture vigorously in a hood, using a micro- 
burner. In 3 to 4 minutes the foam which forms at first will entirely dis- 
appear and the flask becomes filled with dense white fumes. When this 
stage is reached (but no earlier) cover the mouth of the flask with a small 
watch-glass and continue the vigorous heating for 2 minutes. At the end 
of this time, dilute urines will already be green or blue and concentrated 
ones will be a light straw-yellow, the carbonaceous matter being completely 
destroyed. The flame should then be turned low and the gentle boiling pro- 
cess should be continued for 2 minutes, making a total boiling period of 4 
minutes counting from the time the watch-glass was put in place. Remove 
the flame and let the flask cool for 4 to 5 minutes. At the end of 4, or not 
more than 5, minutes, add first 50 c.c. of water, then 15 c.c. of saturated 
sodium hydrate (50 to 55 per cent.) and connect the Kjeldahl flask promptly, 
by means of a rubber stopper and ordinary glass tubing, with a Pyrex Flor- 
ence flask containing 35 to 75 c.c. (depending on the concentration of the 
urine) of o.i N sulphuric acid together with enough water to make a total 
volume of 150 c.c. and a drop or two of alizarin red solution. These deliverv 
tubes are made from glass tubing, small enough to pass into the ready-made 
holes in the rubber stopper. For the sake of flexibility the delivery tube 
must consist of two parts connected with a short piece of rubber tubing. 
When these connections are made, apply the flame again at full force, but 
not directly under the center, until the acid and alkali in the flask have had 
time to mix. The contents in the flask begin to boil almost at once and 4 
to 5 minutes boiling transfers the whole of the ammonia to the receiver. 
The contents in the receiver become heated but the degree is not of sufficient 
moment to cause any trouble. 

After the distillation is complete, titrate the remaining acid in the receiv- 
ing flask against o.i N sodium hydrate, a faint red color being accepted as 
the end-point, if the titration be conducted without previous cooling of the 
fluid. The color deepens on cooling, so that, when time permits, it is more 
satisfactory to cool in running water before titrating. The calculations are 
the same as in the preceding Kjeldahl method. 

This method is not applicable to highly resistant materials, as for example 
milk, which cannot be completely ' destroyed within 6 minutes. Urines 
containing much sugar, also, belong in this class. If fuming sulphuric acid 
be substituted for ordinary sulphuric acid in the preparation of the hydro- 
lyzing reagent, or if 2 c.c. of fuming sulphuric acid are used in addition to 
5 c.c. of the regular reagent, sugar urines are readily destroyed within the 
heating period of 4 to 5 minutes. If this latter modification be used, 25 to 
30 c.c. of saturated sodium hydrate must be added to the digested mixture 
before distilling off the ammonia. 

Folin and Denis' Direct Nesslerization Method. 

This method^ combines the digestion of the urine, as in the preceding 

method, with the direct Nesslerization of the digestion mixture against a 

1 Jour. BioL Chem., 1916, XXVI, 473. See, also, Pearce, Jour. Lab. and Clin. Med., 
1916, II, 130. 



2 26 DIAGNOSTIC METHODS 

known solution of ammonium sulphate. The presence of large amounts of 
sulphates in the digestion mixture was the chief obstacle in the way of this 
method, owing to the precipitation of colored mercury ammonium compounds 
used for the color comparison. For this reason, the results obtained^ by 
earlier workers have not been entirely satisfactory.^ 
Solution and Apparatus Required. 

1. Nessler Reagent. \\ 

Seventy-five grams of potassium iodid are dissolved in 50 c.c. of warm 
distilled water and 100 grams of mercuric iodid are added to this solution. 
Stir for a few minutes, when solution is complete, although it is not usually 
perfectly clear. Dilute this solution with 400 or 500 c.c. of water, filter, and 
make the filtrate up to one liter. This is the stock solution, from which a 
Nessler's solution of any required degree of alkalinity may be made as 
desired. For the determination of nitrogen by this method, the authors 
use a solution of about 2 per cent, alkahnity. To prepare this diluted 
Nessler reagent, proceed as follows: To 300 c.c. of the above double-iodid 
stock solution add 200 c.c. of 10 per cent, sodium hydrate, 500 c.c. of dis- 
tilled water, and mix. (See Non-protein N. of blood.) 

2. Standard Ammonium Sulphate Solution, 

This must be prepared from an absolutely pure salt. The authors state 
that Kahlbaum's ammonium sulphate, labelled c. p. for analysis, is such 
and can be used after drying either over sulphuric acid for a day or two or by 
heating at no degrees C. for an hour before being weighed. 4.716 grams of 
this pure salt are weighed out and dissolved in i liter of 0.2 N solution of 
sulphuric acid (in order to keep out molds). This solution contains i mg. 
of nitrogen per c.c. This solution keeps well and is the stock from which 
weaker solutions are prepared, as desired, by appropriate dilution. For 
use in this method, the standard solution should be of such strength that it 
contains i mg. of nitrogen per 20 c.c, hence the stock solution must be di- 
luted 2c times before being used. 

3. ^ 10 Per Cent, Solution of Sodium Hydrate. 

4. Digestion Mixture, 

A filtered mixture of 100 c.c. of concentrated sulphuric acid, 300 c.c. of 
"syrupy" (85 per cent.) phosphoric acid, and 15 c.c. of 10 per cent, copper 
sulphate solution. 

5. Pi pets and Glass Ware. 

A calibrated long stem i c.c. Ostwald pipet, which will deliver exactly 
I c.c. when drained against the side of the test-tube and then blown clean, 
for measuring the urine; an ordinary i c.c. pipet for measuring the digestion 
mixture; hard glass test-tubes, preferably 190 mm. X 15 mm.; volumetric 
flasks, capacity 100, 200 and 250 c.c; microburners; a high grade colorimeter. 
Technic. 

Dilute the urine so that i c.c contains from 0.7 to 1.5 mg. nitrogen. 

Urines having a specific gravity of 1018 or less should be diluted to one in 

iGulick, Jour. Biol. Chem., 1914, XVIII, 541; Bock and Benedict, Ibid., 1915, XX, 52; 
Taylor and Hulton, Ibid., XXII, 63. 



THE URINE 227 

five. Those of 1030 or over should be diluted to one in twenty. For urines 
ranging between 1018 and 1030 a dilution of one in ten is appropriate. With 
an Ostwald pipet measure into a hard glass test tube i c.c. of diluted urine. 
Add (with an ordinary pipet) i c.c. of the digestion mixture and a piece of 
granite to prevent bumping. Heat the tube over a microburner, with the 
bottom of the test-tube within i cm. of the top of the burner, until nearly 
all the water has been driven off as indicated by the absence of foaming 
and by the appearance of the denser sulphuric acid fumes. This should 
occur in 2 to 5 minutes. Cover the mouth of the test-tube with a watch 
glass and continue the heating with a flame so regulated that only a little of 
the acid fumes escape from the test-tube. In 0.5 to 3 minutes, counted from 
the time the test-tube was closed, the digestion should be clear and blue, 
green, or light straw yellow in color. Continue the heating for another 30 
to 60 seconds, the total heating being in no case less than 2 minutes after 
the test-tube was closed. 

Remove the flame and allow the test-tube to cool for 2 minutes. Add 
water, and rinse the contents of the tube into a 200 or 250 c.c. volumetric 
flask, using about 150 c.c. of water for this purpose. Determine the titratable 
acid content of i c.c. of the digestion mixture (as delivered by the pipet 
regularly used for measuring this solution), using 10 per cent, sodium hydrate 
with phenolphthalein as indicator. Add to the diluted digestion mixture in 
the volumetric flask 10 per cent, sodium hydrate solution in amount equal to 
1 1/8 or 1.375 times the titrating value obtained, plus 2 c.c. for alkalinity. 

Into another volumetric flask, of the same capacity as that used above, 
introduce i c.c. of the digestion mixture and 20 c.c. of the standard ammon- 
ium sulphate solution (the stock solution diluted 20 times, hence 20 c.c. 
contains i mg.). Add about 125 c.c. of water and then the same amount 
of sodium hydrate as in the case of the unknown. Mix well. Now add, 
with a cylinder, to each volumetric flask 15 c.c. of the dilute Nessler's solu- 
tion (see above) and mix quickly. FiU to the mark with water and mix 
thoroughly. Pour out a part of the unknown (as an additional precaution 
against incomplete mixing), and centrifuge, or filter through a small cotton 
plug a portion of the remainder for the colorimetric comparison. If the 
sediment obtained is mixed with a red deposit the Nesslerization has not 
been successful and the determination must be discarded. The liquid 
above the sediment (or the filtrate) must be crystal- clear, not the least bit 
"smoky." 

Adjust the colorimeter, with the standard set at 20 mm. in both cups, 
until the two fields are as nearly alike as it is possible to get them. Then 
make two or three color comparisons with the standard against itself. When 
the result is accurate, replace the standard in one of the cups with the un- 
known and make one leisurely, careful reading. As the standard ammonium 
sulphate solution, as used, contains i mg. in 20 c.c, or 0.05 mg. in i c.c, the 

20 

calculation of the unknown is as follows: r^ ^ :; X 0.0=5 = mg. 

readmg 01 unknown 

nitrogen in i c.c of urine. 



2 28 DIAGNOSTIC METHODS 

(b) Urea (NH2)2CO. 

A discussion of the various factors which have to do with urea excretion 
cannot be taken up at this time. The recent work in the laboratory of Hof- 
meister^ as well as that of Chittenden and of Folin has shown us conclusively 
that it is no longer possible to consider the rate of urea formation as a direct 
measure of protein metabolism. It is without question true, as Leathes^ has 
said, that the nitrogen, or a great part of it, may be removed from the protein, 
converted into urea, and expelled with the urine before the oxidation of the 
rest of the protein molecule has been started upon; and the fact that we can 
trace in the urine excreted in a given time all or the greater part of the nitro- 
gen of the protein taken at a meal, tells us nothing whatever about the fate 
of that part of the protein which contains, it may be, as much as 80 or 90 per 
cent, of the total energy of the protein. Further, urea is not a measure of the 
true protein catabolism, because a great part of it is formed from nitrogen 
that has never been beyond the liver; and it is not the measure of the protein 
energy because it is largely derived from protein by reactions which leave the 
energy value of the molecules from which it originates but little altered. The 
importance of the denitrifying and desamidization reactions of the tissues 
must be much more considered in the future than they have been in the past.^ 

As usually stated in text-books, urea constitutes from 80 to 85 per cent, of 
the total nitrogen output. It has been customary to figure directly the 
amount of tissue protein which must have been decomposed in order to yield 
this amount of urea. These figures can hardly be taken as conclusive of such 
decomposition. Folin has shown that a definite exogenous as well as an en- 
dogenous protein metabolism occurs. With a patient upon a nitrogen-free 
diet, the urea constitutes only about 60 per cent, of the total nitrogen. This 
would represent the true endogenous urea formation. A diet which requires 
the patient to eliminate much higher percentages of urea is, therefore, causing 
increased systemic activity, but is not increasing the direct tissue decomposi- 
tion, as this excess never becomes a part of the system. One of the most 
important laws of protein metabolism is that the amount of nitrogen in the 
body is not increased by, or not in proportion to, an increase in the nitrogen 
intake. 

The amount of urea excreted on an average diet varies from 15 to 40 
grams. ^ Folin finds this excretion to be, on a diet of 119 grams of protein 
yielding 18.9 grams of nitrogen, 298. grams. On a nitrogen-free diet the 
amount of urea is 2.2 grams. This excretion will, of course, vary, depending 
upon the diet. Von Jaksch states that the excretion of urea bears a definite 
relation to the total nitrogen excretion, so that for clinical purposes direct urea 
determinations may well be dispensed with, as the correct urea-content of the 
urine may be found by multiplying the simple nitrogen of the urine by the 

^Lang, Beitrage zur chem. Phys. und Path,, 1904, V, 340. 

2 Problems in Animal Metabolism, Phila., 1906. 

2 See Henriques and Anderson, Ztschr. f. physiol. Chem., 1913, LXXXVIII, 357; Ibid., 
1914, XCII, 21; Davis, Bull. Johns Hopkins Hosp., 1915, XXVI, 154; Hoagland and 
Mansfield, Jour. Biol. Chem., 191 7, XXXI, 487. 

^ See McLean (Jour. Exper. Med., 1915, XXII, 212 and 366) for a discussion of the law 
governing the rate of urea excretion. 



THE URINE 229 

factor 2. This statement should not be regarded seriously by the practitioner 
as the rule, as Folin shows, would require that 93.3 per cent, of the total nitro- 
gen in the urine be in the form of urea. 

Pathologic Variations. 

The pathologic increase in the amount of urea is observed under the same 
conditions as those mentioned under an increase of total nitrogen. Thus in 
febrile conditions, in diabetes mellitus and insipidus,^ after the resorption of 
an exudate, in malignant conditions, and in exophthalmic goiter, the urea 
may be markedly increased. 

In conditions associated with destruction of hepatic parenchyma, or with 
a diminished rate of blood-flow through the liver, the urea excretion may be 
very considerably diminished.^ Thus we find in acute yellow atrophy, 
carcinoma, cirrhosis, and phosphorus poisoning that the normal urea of the 
urine is replaced by other nitrogenous constituents. The normal function of 
the liver in converting ammonium compounds and amino acids into urea is so 
markedly interfered with that the urea may completely disappear from the 
urine in such cases. It should be mentioned that the liver is not wholly 
responsible for the conversion of the ammonia and amino acids into urea, as 
the kidney unquestionably plays some role in this process.^ 

In acute nephritis there may or may not be a diminution in the excretion of 
urea, depending upon the extent of the renal insufl&ciency. In the chronic 
types of nephritis we find that the urea excretion fluctuates to a great extent, 
periods of increase varying with those of decrease. In the early stages, even 
though large amounts of albumin and casts be present, the urea may be 
normal, while in the later stages it is often greatly diminished. 

A diminished excretion of urea is observed in melancholia and in the ad- 
vanced stages of general paresis, while in epilepsy and hysteria an increase or 
a decrease may be observed. In some cases of diabetes mellitus Hirschfeld 
has shown that the urea output may be diminished as the result of delayed 
absorption from the intestine. In these cases of diabetes the ammonia of the 
urine may be markedly increased owing to its combination with acid bodies 
and consequent withdrawal from hepatic activity. 

While urea is a very important substance both clinically and chemically, 
it is very rarely tested by qualitative methods in medical work. The writer 
feels, therefore, that a description of the properties of this substance would 
best be learned by consulting works on physiologic chemistry. 

Determination of Urea. 

The methods for the determination of urea are numerous. Many of them 
are inaccurate although giving results clinically of importance. In the selec- 
tion of a method for the determination of urea one must be governed en- 
tirely by the importance of the urea determination in any specific case. It 

1 See Bassler, Jour. Am. Med. Assn., 1914, LXII, 282. 

2 See Fiske and Karsner, Jour. Biol. Chem., 1913, XVI, 399. 

^ See Folin, Jour. Biol. Chem , 1912, XI, 87; Van Slyke, Ibid., 1913, XVI, 213; Marshall 
and Davis, Ibid., 1914, XVIII, 53; Fiske and Sumner, Ibid,, 285; Jansen, Ibid., 1915, XXI, 
557. Palacios, Am. Jour. Med. Sc, 191 5, CXLIX, 267; Jansen, Nederl. Tijdschr. v. 
Geneesk., 1916, II, 2271; Hammett, Jour. Biol. Chem., 1919, XXXVII, 105. 



230 DIAGNOSTIC METHODS 

should be said in advance that a urea estimation is absolutely useless unless 
taken in conjunction with the total nitrogen. The general practitioner 
usually insists on knowing both the percentage and total amount of urea ex- 
creted with utter disregard both for the nitrogen of the intake and of the 
output. If the urea is of any value at all it should be determined with these 
points in view. The prevailing idea is that a percentage output of two is 
approximately normal and he therefore bases his conclusion upon an increase 
or decrease with this as a standard. Not infrequently single voidings of 
urine are examined for the urea output. Such examinations are worse than 
useless and may even be harmful. 

The methods for the determination of urea are distinctly separable into 
those useful for purely clinical purposes and those for the more exact metabolic 
work. The general practitioner, believing as he does in the importance of 
urea as an indicator of systemic activity and excretion, must have a rapid and 
easy method for estimation of urea. It is to be said, however, that the more 
exact methods would better be applied if reliable conclusions are to be drawn. 

Knop-Hiifner Method. 

The principle of this method is the decomposition of urea by means of 
sodium hypobromite and the measurement of the nitrogen evolved. Sodium 
hypobromite acts upon urea according to the following equation: 

CO(NH2)2+3NaOBr = 3NaBr+2N+C02+2H,0. 

The carbon-dioxide evolved in this reaction is absorbed by the excess of alkali 
used, so that all that is necessary is tomeasure the amount of nitrogen evolved. 
This can be done by direct measurement or by collection in tubes which are 
so calibrated that each c.c. of nitrogen represents a certain percentage of urea. 

Various forms of apparatus, which are termed ureometers, have been 
advised for the estimation of urea by this method. The form introduced by 
Knop and Hlifner is probably the most accurate, but is too complicated for 
general chnical purposes. As this method has absolutely no claim to accuracy 
it is useless in scientific investigations of the nitrogen partition of the urine. 
For clinical purposes, however, it serves as a rough approximation of the 
urea output and is, for this reason, largely used by the practitioner.^ 
|This method, applied as in the following discussion, is given merely because 
of the fact that it is almost the only method of estimating urea which can 
be carried out by the general practitioner. 
Doremus Ureometer. 

This instrument is seen in the accompanying cut. The graduations of the 
tube are such that the number of mg. of urea in the i c.c. of urine used in the 
test are directly read off instead of the number of c.c. of nitrogen formed in 
the^reaction. Aso.oi gram of urea in one c.c. represents i gram per 100 c.c, 
this tube will furnish directly the percentage values of urea. 

1 See Krogh, Ztschr. f . physiol. Chem., 1913, LXXXIV, 370; Robinson and Mueller, Jour. 
Am. Med. Assn., 1914, LXII, 514; Golse (Jour. Pharm. Chim., 1919, XIX, 20) has intro- 
duced a volumetric method based on this test. See, also, Philibert, Ibid., 335, 386 and 434; 
Boyer, Ibid., 346. 



THE URINE 



231 



The tube is filled with a solution of sodium hypobromite made by adding 
I c.c.vof bromin to 40 c.c. of 20 per cent, cold sodium hydrate solution. This 
hypobromite solution decomposes after standing for a few days so that it is 
never wise to attempt to keep such a solution for any length of time. In the 
writer's laboratory a stock solution of 20 per cent, sodium hydrate is prepared 
and the bromin added to it only as occasion requires for preparing fresh solu- 
tions. In this way one will always have the material at hand and need have 
no fear of his stock solution decomposing. After fiUing the tube with this 





Fig. 73. — Doremus ureometer. 



Fig. 74. — Doremus-Hinds ureometer. 

{Hawk.) 



hypobromite solution, i c.c. of urine is added by means of the curved pipet 
accompanying the instrument. In injecting the urine into the solution, the 
curved end of the pipet should be passed well under the curve of the bulb, the 
tube tilted slightly forward, and the urine forced into the hypobromite solu- 
tion with a slow steady pressure. An evolution of gas will be observed at 
once and will cease in a short time. The carbon dioxid given off by the re- 
action of the urea upon the sodium hypobromite is absorbed by the excess of 
alkali and the nitrogen collects in the upper portion of the tube. As soon as 
the evolution of gas has ceased (5 to 10 minutes), the amount of urea is read 
off directly from the calibrations of the tube as previously described. 

In this determination the urine should, theoretically, be free from both 
albumin and sugar. Practically, however, these substances are never re- 



232 



DIAGNOSTIC METHODS 



O^ HiiimH l 



moved as the method yields comparative rather than accurate results. 
Indeed, a very delicate reaction for albumin is given by this test. One may 
be practically certain of the presence of albumin, if the urine, on being in- 
jected into the solution, forms rather characteristic heavy bubbles and if 
a dense froth collects in the upper portion of the tube. This froth settles 
slowly so that the percentage reading requires some time. The forms of this 
apparatus which substitute for the glass foot a wooden base are much to 
be preferred as they are not so easily broken. The modification of this 
instrument, as introduced by Hinds, is seen in the accompanying cut. In 

this form the urine is allowed to run in from 
the smaller graduated tube by opening the 
stop-cock. This modification is an advan- 
tage, but does not yield any more accurate 
results than does the preceding. 
Folin'si Method. 
The principle of this method is as follows : 
At a temperature of i6o°C. crystallized 
magnesium chlorid (MgCl26H20) boils in its 
water of crystallization. If urea be present 
it is decomposed by this boiling solution into 
ammonia and carbon dioxid. If the conver- 
sion be carried out in acid solution, the 
ammonia formed will combine with the acid 
and may then be liberated by alkalinizing 
the mixture. The ammonia is distilled into 
a standard acid solution and may then be 
determined as given under the total nitrogen. 
In this process the preformed ammonia as 
also the trace present in the magnesium 
chlorid will also be determined so that a 
separate estimation of these factors must be 
made and subtracted from the total amount. 
Technic. 
Five c.c. of urine are measured into an 
Erlenmeyer flask of about 200 c.c. capacity, 
5 c.c. of concentrated hydrochloric acid, 20 
grams of crystallized magnesium chlorid, a piece of paraffin about the size 
of a hazelnut and two or three drops of a i per cent, aqueous solution of 
ahzarin-red are added. An especially constructed safety-tube (see cut) is 
then inserted and the mixture boiled until the drops flowing back from the 
safety-tube produce a very perceptible bump or hissing sound on coming in 
contact with the solution (10 to 15 minutes). The temperature^ is then 

^Ztschr. f. physiol. Chem., 1901, XXXII, 504; Ibid., 1903, XXXVII, 548. 

- Folin advises (Handb. d. Biochem. Arbeitsmeth., 191 1, V, 286) the use of small glass 
bulbs containing solid HgClI as an indicator of the proper temperature of this reaction. 
This substance melts at i53°C., which is the optimum temperature to insure complete de- 
composition of the urea. 




Fig. 



75. — FoHn's urea apparatus. 
{Hawk.) 



THE URINE 233 

somewhat reduced and the heating continued for one hour. It is important 
in this process that the reaction must not remain alkahne, and, therefore, 
as soon as the material in the flask turns red a very few drops of the acid 
distillate in the safety- tube are shaken back into the flask. ^ At the end 
of the hour the contents of the flask are washed into a liter Kjeldahl flask 
with about 700 c.c. of water. Twenty c.c. of 10 per cent, sodium hydrate 
are then added and the ammonia distilled, as described under total nitrogen, 
into a standard tenth-normal solution of sulphuric acid. This distillation 
should be continued until the contents of the liter flask are nearly dry or 
till the distillate shows no trace of ammonia with litmus paper. This will 
require about one hour. The distillate is then boiled to drive off the 
carbonic acid, is then cooled, and titrated with tenth-normal sodium hydrate 
to determine the amount of acid which combined with the ammonia formed. 
Alizarin red or methyl red are used as indicators. One c.c. of tenth- 
normal sulphuric acid is equivalent to 0.001704 gram of ammonia (NH3), 
or 0.001401 gram of nitrogen. If the nitrogen value be multiplied by 2.143 
the result will be the amount of urea in the 5 c.c. of urine taken. From 
the total c.c. of n/io sulphuric acid neutralized must be subtracted the 
n/io sulphuric acid values for the preformed ammonia as well as for the 
ammonia which may be present as an impurity in the 20 grams of magnesium 
chlorid used. 

It has been found by Schoorl that when carbohydrates and urea are 
heated together they form very stable condensation products (ureids). For 
this reason this method of Folin does not give accurate results with saccharin 
urine. A combination of this method with that of Morner, which will be 
described later, will give absolutely accurate results. In the determination 
of the ammonia values of the preformed ammonia and of the magnesium 
chlorid, the later methods of Folin must be used. 
Momer-Sjoqvist Method. 

This method^ is an extremely accurate one, but no more so than that of 
Folin, except in saccharin urines. If albumin be present it must, however, 
be removed by heat and acetic acid and the original volume of the urine re- 
stored. If the urine contains large amounts of hippuric acid this method 
may not give accurate results, as Salaskin and Zaleski have shown. 
Technic. 

Five c.c. of urine are placed in a flask with 5 c.c. of baryta mixture con- 
sisting of a saturated barium chlorid solution containing 5 per cent, of barium 
hydrate. One hundred c.c. of a mixture of two parts of 97 per cent, alcohol 
and one part of anhydrous ether are then added and the mixture allowed to 
stand in a closed flask overnight. It is then filtered and the residue washed 
with fresh alcohol and ether mixture and the combined filtrates evaporated 
at a low temperature (6o°C.). Urea will be practically the only nitrogenous 
body left in solution, with the exception of traces of ammonia. When the 
evaporated filtrate has been reduced to about 25 c.c. in volume, a few c.c. 

1 See Folin, Jour. Biol. Chem,, 1912, XI, 507. 

2 Skand. Arch. f. Physiol., 1891,^11, 438; Ibid., 1903, XIV, 247. 



234 DIAGNOSTIC METHODS 

f 

of water and a small amount of calcined magnesium oxid are then added, 
the mixture stirred, and heated to drive off the ammonia; or the residue may 
be treated by Folin 's method as previously described. This heating is con- 
tinued until the vapor shows no alkalinity when tested with moistened litmus 
paper, a result usually obtained when about lo to 15 c.c. of the mixture remain. 
The fluid and the residue are then washed into a Kjeldahl flask and treated 
with concentrated sulphuric acid, copper sulphate, and potassium sulphate 
as in the determination of total nitrogen. One part of nitrogen is equivalent 
to 2.143 grams of urea. 

If much hippuric acid be present, Braunstein^ advises oxidation of the 
urea by heating the evaporated residue with 10 grams of solid phosphoric 
acid (in an air-bath) to 140 to i45°C. for five hours. Alkalinize and distill 
as previously described. Benedict's^ newer method also yields accurate 
results. 

Urease Method. 

Marshall has introduced a method for the determination of urea in various 
body fluids, which is based on an entirely different principle from the others 
advocated and which is accurate and extremely simple.^ He takes advantage 
of a fact, first discovered by Takeuchi, that an aqueous extract of soy bean 
{glycine hispida) contains a ferment which converts urea into ammonium 
carbonate and which neither acts upon anything else nor is interfered with by 
anything ordinarily found in the various body fluids. In his method Mar- 
shall employs this aqueous extract, but this has the disadvantage of requiring 
many hours to react quantitatively and, further, of losing its activity on 
standing for a few days, the loss being more rapid the higher the temperature 
at which it is kept. For these reasons the writer prefers to adopt the modifica- 
tion of Van Slyke and Cullen. These workers have produced a permanent 
preparation of this ferment by the following method: Extract soy bean meal 
with 5 parts of water and pour this extract into 10 volumes of acetone. Filter 
off the precipitate and dry it. ^ 

This urease dissolves readily in water, forming an opalescent solution. 
''Under given conditions, a given amount of it will decompose a definite 
amount of urea per minute and no more, regardless of how much excess urea 
may be present. The preparation is most active at a temperature approxi- 
mately 55°C. (i3i°F.) and in a perfectly neutral solution. Between 10 and 
5o°C. (50 and i2 2°F.), increasing the temperature over any 10° interval 
doubles the rate at which the preparation acts. Heavy metals, or acid in a 

iZtschr. f. physiol. Chem., 1901, XXXI, 381. 

2 Jour. Biol. Chem., 1910, VIII, 405. See, also, Milroy, Biochem. Jour., 1913, VII, 399. 

3 Takeuchi, Jour. College Agriculture, Tokio, 1909, 1, i; Chem. Ztg., 191 1, XXXV, 408; 
Marshall, Jour. Biol. Chem., 1913, XIV, 283; Ibid., 1913, XV, 495; Hahn and Saphra, 
Deutsch. med. Wchnschr., 1914, XL, 430; Van Slyke and Cullen, Jour. Am. Med. Assn., 
1914, LXII, 1558; Jour. Biol. Chem., 1914, XIX, 141 and 211; Van Slyke, Zacharias and 
Cullen, Deutsch. med. Wchnschr., 1914, XL, 1219; Plimmer and Skelton, Biochem. Jour., 
1914, VIII, 70; Hahn, Deutsch. med. Wchnschr., 1915, XLI, 134; Eigenberger Ztschr. f. 
physiol. Chem., 1915, XCIII, 370; Jacoby and Sugga, Biochem. Ztschr., 1915, LXIX, 116. 

•* If one does not wish to prepare this urease for himself, he may obtain it from the Ar- 
lington Chemical Company of Yonkers, N. Y.; Mateer and Marshall, Jour. Biol; Chem., 
1916, XXV, 297, have shown that the jack bean (Canavalia ensiformis) is about 15 times 
richer in urease than is the soy bean. 



I 

I 



THE URINE 235 

relatively slight concentration, destroy the activity. The urease is not quite 
so sensitive toward an alkaline reaction, but its activity is decreased by even 
weak bases. Ammonium carbonate depresses the activity to about one-eighth 
the optimum, so that the action of the enzyme is self -retarding.^ The retard- 
ing effect of the ammonium carbonate formed can be neutralized and 
conditions for optimum activity obtained by the addition of potassium di- 
hydrogen phosphate and dipotassium hydrogen phosphate in equimolecular 
proportions. This phosphate mixture also acts as a stabilizer to solu- 
tions of enzyme." 

Preparation of Enzyme Solution. 

Two grams of the enzyme preparation, 0.6 gram of dipotassium hydrogen 
phosphate and 0.4 gram of potassium dihydrogen phosphate are stirred up 
with a rod in 10 c.c. of water. The preparation dissolves quickly, forming an 
opalescent solution. If this solution be covered with toluol it will hold its activ- 
ity for a week or so, but it is better technic to employ relatively fresh solutions. 
Technic. 

This method employs a portion of the technic originally introduced for 
determination of ammonia by Folin (see page 240). Dilute 5 c.c. of urine 
with water to 50 c.c. Mix this thoroughly and transfer, by means of a pipet, 
5 c.c. of this diluted urine to a clean, fairly heavy test-tube of about 50 c.c. 
capacity. Add i c.c. of the enzyme solution and i drop of caprylic alcohol 
or a few drops of benzol to prevent foaming during the later aeration. Close 
this tube (A) with a stopper and allow it to stand 1 5 minutes in order to per- 
mit the enzyme to act upon the urea present. 

Into a second test-tube (B) measure with a buret 25 c.c. of fiftieth normal 
(N/50) hydrochloric or sulphuric acid and add i drop of a i per cent, solution 
of alizarin as an indicator and i drop of caprylic alcohol or benzol. 

By means of glass and rubber tubing connect these two tubes in such a 
way that air may be forced through tube A into tube B (see page 240). After 
tube A has stood for 15 minutes, pass the air current for about }4 minute in 
order to carry over into the acid any ammonia which may have passed into 
the upper portion of tube A. Now shut off the air, open tube A and pour into 
it 4 or 5 grams of dry potassium carbonate. ^ Close tube A at once and pass 
the air current till all the ammonia is driven into tube B. This time depends 
on the rate at which the air is passed. With the ordinary apparatus this 
varies from 15 to 30 minutes. 

When this process is finished, titrate the excess of acid in tube B with 
fiftieth normal (N/50) sodium hydrate, titrating to the appearance of the first 
permanent red point. Subtract the number of c.c. of standard alkali used 
from 25 (the number of c.c. of standard acid originally measured into tube 
B) and multiply this result by 0.056 to obtain the grams of urea nitrogen plus 
ammonia nitrogen in 100 c.c. of urine. 

1 See Falk, Jour.Biol. Chem., 1917, XXVIII, 389; Van Slyke and Cullen, Ibid., 391. 

2 Fiske, Jour. Biol. Chem., 1915, XXIII, 455, advises the use of 5 c.c. of an alkaline 
solution prepared as follows: Dissolve 500 grams K2CO3 in 500 c.c. of water with slight 
heating; add 10 c.c. of a 30 per cent, solution of potassium oxalate, filter and allow to cool. 
This is concurred in by Van Slyke and Cullen, Jour. Biol. Chem., 1916, XXIV, 117. 



236 DIAGNOSTIC METHODS 

The ammonia nitrogen, which is not referable to the decomposition of urea, 
is obtained by the same method as the above, using 5 c.c. of undiluted urine 
and adding no urease solution. In this latter case multiply the number of c.c. 
of excess acid by 0.0056 instead of 0.056 to determine the percentage of 
ammonia nitrogen originally present in the urine. Subtract this latter 
figure from the figure for the combined nitrogen of urea and ammonia and 
obtain the true figure for the ammonia nitrogen derived from the action of 
the urease. If this nitrogen figure be multiplied by 2.143, one obtains the 
percentage of urea present as such.^ 

Folin and Youngburg^s Direct Nesslerization Method. 

This method^ is a modification of the method of Folin and Denis^ made 
necessary by the fact that Merck's blood charcoal, used in the latter method, 
is difficultly obtainable at the present time. It is a combination of the 
urease method just discussed with that of direct Nesslerization, as treated 
under Nitrogen, with some modifications. Instead of the charcoal, these 
authors use "Permutit," an aluminate silicate, zeolite, discovered by Gans.^ 
Technic. 

Wash about 3 grams of permutit in a flask with 2 per cent, acetic acid' 
then twice with water; add 5 grams of fine jack bean meaP and 100 c.c. of 
30 per cent, alcohol. Shake gently but continuously for 10 to 15 minutes 
and filter. The filtrate contains practically the whole of the urease and 
extremely little of other materials. Add i c.c. of this urease solution to 
I c.c. of diluted urine (dilution usually 1:10) in a stoppered test-tube and 
digest in a beaker of warm water (40-55 degrees C.) for 5 minutes or at room 
temperature for 15 minutes. As a buffer mixture, add a drop of sodium 
pyrophosphate solution (140 grams of sodium pyrophosphate and 20 grams 
of glacial phosphoric acid per liter) at the beginning of the digestion. At 
the end of the digestion period, transfer the contents of the test-tube to a 200 
c.c. volumetric flask, diluting to a volume of about 150 c.c. 

To another 200 c.c. volumetric flask add i mg. of N in the form of am- 
monium sulphate (see Nitrogen Determination for the method of preparing 
this solution) ; to this standard add i c.c. of the urease solution and dilute 
to about 150 c.c. 

To the solutions in each volumetric flask add 20 c.c. of the dilute Nessler 

solution discussed under Nitrogen or, on account of certain impurities in the 

mercuric iodid obtainable on the market, prepare the Nessler solution as 

follows:^ Transfer 150 grams of potassium iodid and no grams of iodin to a 

^ Instead of using the method of aeration, one may titrate directly the ammonium car- 
bonate formed by the action of the urease on the urea, using somewhat larger amounts of 
urine (5 c.c.j and titrating with N/io hydrochloric acid, with methyl-orange as an indicator. 
This was the original method of Marshall, but it does not yield as accurate results as the 
technic described. 

2 Jour. Biol. Chem., 1919, XXXVIII, 11 1; see, also, Sumner and Bodansky, for a fur- 
ther Nesslerization method. Ibid., 57. 

3 Jour. Biol. Chem., 1916, XXVI, 501. 

^ Jahrd. k. preuss. Geol. Landesanstalt, 1905, XXVI, 179; 1906, XXVII, 63. This 
substance is obtainable from the Permutit Company, 30 E. 42nd St., New York. 

^ This meal, as also urease from the soy bean, may be obtained from the Arlington 
Chemical Company, Yonkers, N. Y. 

6 Folin and \Vu, Jour. Biol. Chem., 1919. XXXVIII, 89. 



THE URINE 237 

500 c.c. Florence flask; add 100 ex. of water and an excess (140 to 150 grams) 
of metallic mercury. Shake the flask continuously and vigorously for 7 to 
15 minutes or until the dissolved iodin has nearl}^ disappeared. The solu- 
tion becomes quite hot. When the red iodin solution has begun to become 
visibly pale, though still red, cool in running water and continue the shaking 
until the reddish color of the iodin has been replaced by the greenish color 
of the double iodid. This whole operation usually does not take more than 
15 minutes. Now separate the solution from the surplus mercury by decan- 
tation and washing with liberal quantities of distilled water. Dilute the solu- 
tion and washings to a volume of 2 liters. This is the stock Nessler solution, 
from which the dilute form is prepared as follows: Introduce into a large 
bottle 3500 c.c. of ic per cent, sodium hydrate solution, add 750 c.c. of the 
double iodid stock solution and 750 c.c. of distilled water. 

After the addition of the dilute Nessler solution, dilute the mixture in 
the volumetric flasks to volume and make the color comparisons in the colori- 
meter, as discussed under Nitrogen. The ammonia nitrogen, due to the 
preformed ammonia of the urine is, of course, included in the figure obtained 
and must be determined, as outlined later, and deducted from this figure 
in order to arrive at the exact nitrogen value referable to urea. 

The authors call attention to the fact that there are many kinds of 
biological nitrogenous materials, particularly amino-acids, peptones, and 
albumins, which prevent the development of color reactions given by am- 
monia and Nessler's reagent. The color in such cases tends to be visibly 
more greenish and less distinctly red than, the standard. Correct results 
are, however, given by this method even with albuminous urines. Further 
attention is called to the fact that the glass ware use'd in this test must be 
washed thoroughly with nitric acid or, as others advocate, with potassium 
iodid and alkali, in order to remove traces of mercury compounds which may 
adhere to the glass. 

(c) Ammonia (NH3). 

This substance, although chemically belonging in the class of inorganic 
compounds, is so closely related to the nitrogen rnetabolism that it is more 
properly discussed under the heading of Nitrogenous Bodies. 

Ammonia is one of the most important products of protein metabolism. 
It is constantly present in small amounts in normal urine averaging about 
0.85 gram of NII3 in 24 hours, representing from 4 to 5 per cent, of the total 
nitrogen. It is present in combination with various acids and may repre- 
sent largely a portion of the nitrogen which has not been transformed into 
urea, but has been used to combine with acid substances formed in the 
protein metabolism of the body. Any increase in the production of acid 
in the system or any increased intake of noncarbonate-forming acids will 
lead to an increased excretion of ammonium salts. This is an important 
factor in the metabolism of conditions associated with an acidosis. 

The total output of ammonia will vary under normal conditions with 

the diet or, in other words, with the intake of total nitrogen. ^ While the 

1 See Tanji, Deutsch. Arch. f. klin. Med., 1914, CXVI, 92; Wills and Hawk, Jour. Am. 
Chem. Soc, 1914, XXXVI, 158, have shown an increased output of ammonia following 
and proportional to increased ingestion of water. 



238 DIAGNOSTIC METHODS 

increase of the total nitrogen of the urine or increased nitrogen intake is 
largely in the form of urea, yet a small increase in the absolute amount of 
ammonia must occur. Likewise we observe a diminished intake of nitrogen 
reducing the absolute value of ammonia, but largely increasing its relative 
value. Thus Folin finds with a total excretion of 16 grams of nitrogen, an 
ammonia output of 0.85 gram (4.3 per cent.) ; while on a nitrogen-free diet a 
total nitrogen output of 3.6 grams was observed with an ammonia elimination 
of 0.51 gram (11. 3 per cent.). We therefore conclude with Folin as follows: 
''With pronounced diminution in the protein metabolism (as shown by the 
total nitrogen in the urine), there is usually, butjQot always, and therefore not 
necessarily, a decrease in the absolute quantity of ammonia eliminated. A 
pronounced redaction of the total nitrogen is, however, always accompanied 
by a relative increase in the ammonia nitrogen, provided that the food is not 
such as to yield an alkaline ash." 

Although the ammonium salts of many organic acids are converted into 
urea in the system we find the ammonium salts of the sulphuric and phos- 
phoric acids formed in the decomposition of protein material are excreted as 
such. Moreover, we observe that an increased consumption of fat, either 
taken in as food or derived from the tissue, is associated with a combination 
of ammonia with the fatty acids. This provision of metabolism, by which the 
system is protected against the deleterious effects of increased acidity by 
neutralization of acid compounds with ammonia, is of the greatest impor- 
tance, as the fixed alkalies of the tissues are thereby maintained in their 
usual concentration unless the pathologic processes be extreme. 

Pathologic Variations. 

An increased output of ammonia is observed in cases of diminished oxida- 
tive powers of the system, in febrile diseases, in hepatic disturbances such as 
carcinoma and acute yellow atrophy, in uremia, in acid intoxication, in 
dyspnea from any cause, in the toxic vomiting of pregnancy, in delayed chloro- 
form poisoning, and especially in diabetes mellitus. In this latter condition 
the degree of acidosis may be conveniently followed by watching the 
ammonia output. 

A reduction in the amount of ammonia is observed in many cases of 
nephritis and in some cases of carcinoma of the stomach, although there 
is at the same time a diminished excretion of hydrochloric acid in the gastric 
contents. Edsall reports a reduction in cases of periodic insanity preceding 
the attack, while a rise is observed as the attack proceeds. Administration 
of large doses of the fixed alkalies will usually diminish the ammonia output.^ 

Quantitative Determination of Ammonia. 

Like the methods given under Urea, many have been advanced for the 
determination of the urinary ammonia. One of these, though inaccurate, 
has been so long used and even to-day is so relied upon in many quarters that 

^ See Janney, Ztschr. f. physiol. Chem., 191 2, LXXVI, 99; Palmer and Henderson, 
Arch. Int. Med., 1915, XVI, 109; Denis and Minot, Jour. Biol. Chem., 1918, XXXV, loi; 
Marriott and Rowland, Arch. Int. Med., 1918, XXII, 477. 



THE URINE 



239 



the writer includes it with the understanding that he advocates only the ac- 
curate methods. If the ammonia is worth determining, definite results 
should be sought and hence the most accurate methods are the ones to be used. 
They are no more complicated, not as time-consuming, and give more reliable 
results. 

Method of Schlosing. 
This method is the one most commonly used, but is open to the objection 
that it does not yield accurate results and is time-consuming, but has thv 
advantage of simplicity. It is argued by many that the element of time is of 
no importance, as clinically one would not wait for an ammonia determination 
before instituting vigorous treatment. On the other hand, in metabolic work 
it is of a great advantage to get the work out of the way as quickly as is 
possible and consistent with accurate results. 

Technic. 

Twenty-five c.c. of urine are placed in the vessel B (preferably a Petri 
dish) (see cut). Above this is placed a glass triangle upon which rests a dish 




Fig. 76. — Schlosing's ammonia apparatus. 

(C) containing 20 c.c. of tenth-normal sulphuric acid. Twenty c.c. of milk 
of lime are then poured into the dish containing the urine and the whole cov- 
ered with a bell-jar, the borders of which have been well greased to make an air- 
tight union when the jar is placed upon the glass plate. This apparatus 
is then allowed to stand at room temperature from four to five days, during 
which time the ammonia, liberated by the action of the milk of lime upon 
the ammonium salts of the urine, will be taken up by the sulphuric acid in 
the vessel C. At the end of this time the bell-jar is removed, the acid titrated 
with tenth-normal sodium hydrate, and the number of c.c. of remaining acid 
determined. One c.c. of tenth-normal sulphuric acid neutralized by the 
evolved ammonia represents 0.001704 gram of ammonia. This figure is 



240 



DIAGNOSTIC METHODS 



multiplied by 4 to obtain the percentage ammonia value. If any moisture is 
present on the inside of the bell-jar it should be washed into the sulphuric 
acid before titration. 

This method, as previously stated, does not give accurate results owing 
to the fact that ammonia may be split off from urea and thus give figures 
which are somewhat high. It has been found that if the apparatus be kept 
at 38°C., the time necessary for this reaction may be reduced to 48 hours. 
If we add to the urine instead of the milk of lime, 0.5 gram of sodium carbon- 
ate and about 10 grams of sodium chlorid, no ammonia will be split off from 
the urea and no decomposition of the urine will occur (Schaffer). 




Folin's ammonia apparatus. {Hawk.) 



Felines Method. 

The ammonia in this method^ is set free by the addition of a weak alkali 
(sodium carbonate), is then removed from the urine at ordinary room tem- 
perature by means of a strong air-current, is collected in tenth-normal sul- 
phuric acid and then titrated. 
Technic 

Twenty-five c.c. of urine are poured into an aerometer cylinder (30 to 40 
cm. high), 15 grams of potassium oxalate, ^ i gram of dry Na-iCOs and some 
crude petroleum (to prevent foaming) are added. The upper end of the 
cylinder is then closed by means of a doubly perforated rubber stopper, through 
which pass two glass tubes, only one of which is long enough to reach below the 
surface of the liquid. ^ The shorter tube (about 10 cm.) in length is connected 

^Ztschr. f. physiol. Chem., 1902, XXXVII, 161; Ibid., 1903, XXXIX, 477. 

'^ Jour. Biol. Chem., 1910, VIII, 497; Denison, Jour. Lab. and Clin. Med., 191 7, II, 204. 

2 Folin and Macallum (Jour. Biol. Chem., 191 2, XI, 523) have originated a micro-chem- 
ical method, which permits of much quicker estimation, i to 5 c.c. of urine, depending on 
the amount suspected, are placed in a large Jena test-tube by means of an Ostwald pipet. 
Add to the urine a few drops of a solution containing 10 per cent, potassium carbonate and 
15 per cent, potassium oxalate and follow this with a few drops of kerosene or benzol to pre- 
vent foaming. Pass the strong air current for 10 minutes, collecting the ammonia in a 100 
c.c. volumetric flask containing about 20 c.c. of water and 2 c.c. of N/io hydrochloric acid. 
Nesslerize this solution against a standard ammonium sulphate solution as described in 
section on Blood or titrate it with N/50 alkali using methyl-red as an indicator. 



THE URINE 



241 



with a calcium chlorid tube filled with cotton, which in turn is connected 
with a glass tube extending to the bottom of a wide-mouthed bottle (capacity 
about 500 ex.) which contains 20 c.c. of tenth-normal sulphuric acid, 200 c.c. 
of water and a few drops of an indicator (alizarin-red). The complete ab- 
sorption of the ammonia by the sulphuric acid is most easily insured by the 
use of a simple absorption tube which compels a very intimate contact of the 
air coming from the cylinder with the acid and water in the absorption bottle. 
This absorption bulb consists of a glass tube, measuring about 8 mm., in 
diameter, one extremity of which has been blown into a small bulb. By 
means of a heated platinum wire, 10 to 12 holes, each about i mm. in diameter, 
are made in this bulb. 

The absorption bottle is then attached by means of a glass tube and 
rubber connection to a filter pump which will draw 700 liters of air per hour.^ 
The air passing through the alkaline urine will draw the ammonia into the 
standard acid in from one and one-half to two hours. In order to exclude 
any error due to the presence of ammonia in the aspirated air, 
a similar absorption apparatus is attached to the distal side of 
the evolution flask. The amount is then determined by titra- 
tion of the standard acid with tenth-normal sodium hydrate, 
using methyl-red as an indicator. Steel, Gies and others advise 
the use of NaOH instead of NasCOs in this method on the 
ground that the latter will not liberate NH3 from ammonium 
magnesium phosphate. This would not seem to militate 
against the method as the triple phosphate is rarely, if ever, 
present in the urine in appreciable amounts except when the 
urine is decomposed. Under such circumstances the ammonia 
factor would be useless from the metabolic standpoint. 

Folin and Bell's Direct Nesslerization Method. 

This method^ is a modification of that of Folin and Denis^ 
in which permutit is used to absorb the creatinin, which 
interferes with the Nesslerization process, instead of the 
Merck's blood charcoal, which is almost nonobtainable. The 
removal of am.monia by this mineral is not an adsorptive yig 78 — 
phenomenon. The chemical affinity of the active group in Folin's absorp- 
the reagent for ammonia is remarkably strong, so that under (^^.^^ ) 
suitable conditions the exchange becomes quantitative as far as 
the ammonia is concerned. This absorption is best from neutral solutions, 
but is also good from weakly acid solutions. The presence of much acid 
is not good because the reagent is dissolved by acids. In alkaline 
solutions ammonia is not absorbed. The serviceability of the reagent 
depends, fundamentally, on the fact that in the presence of sodium hydrate 



^ In laboratories in which compressed air is available, the ammonia may be forced from 
the generating cylinder into the cylinder containing the standard acid. In this case, a 
cylinder containing 100 c.c. of 10 per cent, sulphuric acid is interposed between the air blast 
and the generating cylinder in order to catch any ammonia which may be present in the air. 

2 Jour. Biol. Chem., 1917, XXIX, 329. 

3 Ibid., 1916, XXVI, 497; see, also, Sumner, Ibid., 1918, XXXIV, 37. 

16 



242 DIAGNOSTIC METHODS 

the absorbed ammonia is again set free. This reagent does not appreciably 
deteriorate by being used. After washing away the Nesslerized ammonia 
and surplus alkali first with water, then with one portion of 2 per cent, 
acetic acid, then once more with water, the powder remaining is efficient 
and usable. 

Technic. 

Transfer about 2 grams of the permutit (either fresh or well washed, as 
above) to a 200 c.c. volumetric flask. Add about 5 c.c. of water (no more) 
and with an Ostwald pipet introduce i or 2 c.c. of urine, or with a 5 c.c. 
pipet introduce 5 c.c. of previously diluted urine (corresponding to i or 2 
c.c. of original urine). Rinse down the added urine by means of a little 
water (i to 5 c.c.) and shake gently but continuously for 5 minutes. Rinse 
the powder to the bottom of the flask by the addition of water (25 to 40 c.c.) 
and decant. Add water once more and decant. (In the case of urines rich 
in^bile it is advisable to wash once or twice more.) Add a little water to the 
powder, introduce 5 c.c. of 10 per cent, sodium hydrate solution, mix, then 
add more water until the flask is about three-fourths full. Shake for a few 
seconds and then add 10 c.c. of Nessler's reagent (prepared as directed 
under total nitrogen and urea). Mix and let stand for 10 minutes or longer. 
Fill up to the mark with water, miix, and make the color comparison in the 
colorimeter with the standard ammonium sulphate solution. This standard 
solution is prepared as directed under total nitrogen and should be, in this 
case, I mg. or, less frequently, 0.5 mg. of ammonia nitrogen, Nesslerized as 
above with the addition of 5 c.c. of 10 per cent, sodium hydrate solution and 
10 c.c. of the dilute Nessler's solution, diluted to 200 c.c. 

Formalin Method. 

This method, originated by Ronchese^ and Malfatti,^ depends on the 
fact that a solution of an ammonium salt, treated with formaldehyd, decom- 
poses with the formation of hexamethylen-tetramin, the acid combined with 
the ammonia being liberated. This can then be determined by titration. 

Dilute 10 c.c. of urine with 50 c.c. of water, add 2 or 3 drops of a i 
per cent, alcoholic solution of phenolphthalein and neutralize with N/io 
NaOH. Five c.c. of formalin, previously neutralized with N/ 10 NaOH, are 
added to the neutralized urine and the mixture is again titrated with N/io 
NaOH to the appearance of a faint, permanent pink color, the amount of 
alkali used being noted. It is evident that i c.c. of this N/io NaOH is equiva- 
lent to I c.c. of N/io NH3 or, in other words, represents 0.001704 gram of 
NH3. Multiply the number of c.c. of N/io NaOH used by this factor and 
obtain the NH3 in 10 c.c. of urine. 

(d) Uric Acid, C5H4N4O3. 

In times past uric acid has been credited with* much more clinical im- 
portance than is to-day ascribed to it. It is not, as many believe, a product 
of protein decomposition, as such, but can be derived only by the splitting of 

^ Jour. Pharm. et Chim., 1907, XXV, 611; Bull. Soc. Chim. de France, 1907, I, 900. 
2Ztschr. f. anal. Chem., 1908, XL VII, 273. 



THE URINE 



243 



the nucleo-proteins. These nuclein bodies are compounds of protein with 
nucleic acid, the latter constituent splitting up into thymic acid and deriva- 
tives of "purin," among which we find uric acid, xanthin, hypoxanthin, etc. 
Uric acid, as well as the purin bases to be discussed later, are derivatives 
of Fischer's hypothetical purin nucleus, with the formula C5H4N4 and the 
following graphic structure. The figures in brackets represent the number 
of the atom or group of atoms : 



(i) 



N= 



(2) HC 



(3) N- 



:CH 

i 
(5) 

C- 



(4) 

-c- 



(6) 

(7) 
NH 

\ 
CH 

(9)/' 

— N 



(8) 



Each of the purin derivatives is formed by the replacement of one or 
more of the hydrogen atoms in this nucleus by various atoms or groups of 
atoms. Thus we find that uric acid is a derivative in which 3 atoms of 
oxygen have been substituted for the hydrogens in positions 2, 6, and 8. The 
hydrogen atoms are not replaced, but shift in the direction of the double 
bonds, passing to the nitrogen atoms 1,3, and 9. The structure of uric acid 
with the formula C5H4N4O3 is 



HN 



OC 



HN 



CO 



NH 



\ 
CO 

/ 



C NH 

Its chemical name is, therefore, 2, 6, 8, tri-oxy purin. 

Uric acid is derived from the nucleins of the food, as well as from the 
nucleins of the tissues.^ We can conceive, consequently, that we may have a 
uric acid excretion which may be, in part, referable to each of these factors. 
This is the basis of the theory of endogenous and of exogenous purin metabo- 
lism, the former representing the uric acid formation from the tissue nucleins, 
the latter that from the food directly. It is self-evident that the exogenous 
purin metabolism, being directly dependent upon the diet, will vary more 
than will the endogenous form. This latter type is dependent not only upon 
the metabolism of the various cellular elements of the body, but also upon the 
degree of direct synthesis of uric acid in the system, as well as upon the extent 
of conversion of uric acid into urea. This latter statement must not be inter- 
preted as meaning that uric acid is simply a stage in the conversion of all 
protein into urea. This idea, which held sway for so long, has fortunately 

^ See Thannhauser, Ztschr. f. physiol. Chem., 1914, XCI, 32Q; Thannhauser and 
Boomes, Ibid., 336; Bass, Arch. f. exper. Path. u. PharmakoL, 1914, LXXVI, 40; Taylor and 
Rose, Jour. Biol. Chem., 1914, XVIII, 519; Morris, Ibid., 1916, XXV, 205. 



244 DIAGNOSTIC METHODS 

been abandoned. However, we do know, from the work of Frerichs 
and Wohler that the system does transform a certain amount of uric acid 
into urea. 

Burian and Schur point out that the uric acid ehminated by man on 
a purin-free diet (endogenous uric acid metaboHsm) is for each individual 
a constant quantity and entirely independent of the total amount of nitrogen 
eliminated. This fact has so firmly fixed itself in the minds of the profes- 
sion that the important results of Folin have been largely unnoticed. This 
latter worker finds that "when the total amount of protein metabolism is 
greatly reduced, the absolute quantity of uric acid is diminished, but not 
nearly in proportion to the diminution in the total nitrogen, and the per cent, 
of the uric acid nitrogen in terms of the total nitrogen is, therefore, much in- 
creased." It would seem, therefore, that this point is still very debatable. 
Quoting again from Folin "if the endogenous uric acid is to be considered as 
derived from the cell nucleins exclusively, it would, indeed, seem highly plaus- 
ible that the quantity should tend to remain constant, even with very great 
variations in diet. Rigid proof that the endogenous uric acid elimination 
is for each individual a constant quantity would be strong evidence in favor 
of such a theory. Burian and Schur support the view that the endogenous 
uric acid is derived from the cell nucleins, but they contend that in man about 
one-half of the uric acid so derived is destroyed inside the organism and that 
only the other half is eliminated. With the introduction of this important 
modification of the nuclein theory, there is no longer any reason why the uric 
acid elimination should not be a decidedly variable factor which might well 
be susceptible to change under the influence of many different changes in the 
conditions, among others, changes in diet." It will be seen, therefore, that one 
must be on his guard in drawing conclusions from the amount of uric acid in 
the urine, as to the degree of nuclein decomposition within the system.^ 

Physiologic Variations. 

The output of uric acid varies, depending upon the diet, from 0.2 to 2 
grams in 24 hours. On a diet of 119 grams of protein with a total urinary 
nitrogen output of 16 grams the uric acid eliminated was 0.37 gram (0.8 per 
cent, of total N); while on a nitrogen-free diet with a urinary nitrogen of 3.6 
grams the output of uric acid was 0.09 gram (2.5 per cent, of total N). It is 
increased physiologically by increase in the nucleins of the diet, sweet-breads, 
liver, kidneys, and brain yielding very large amounts of uric acid. A meat 
diet will lead to a larger excretion of uric acid than will a vegetable diet, the 
maximum output being observed about five hours after a hearty protein meal. 
According to Horbaczewski, this increase is dependent upon the leucolysis 
which occurs at the time of the disappearance of the digestive leucocytosis. 

1 See Siven, Arch. f. d. ges. Physiol, 191 2, XCLV, 283; Ibid., 1914, CLVII, 582; Faustka, 
Ibid., 1914, CLV, 523;Raiziss, Dubin and Ringer, Jour. Biol. Chem., 1914, XIX, 473; Daniels 
and McCrudden, Arch. Int. Med., 1915, XV, 1046; von Moraczewski and Herzfeld, Ztschr. 
f. klin. Med., i9i5,LXXXII, 61; Umeda, Biochem. Jour., 1915. IX, 421; Mendel and Stehle, 
Jour. Biol. Chem., 1915, XXII, 215; Fine, Ibid., XXIII, 471; Wells, Ibid., 1916, XXVI, 
319; Neuwirth, Ibid., 1917, XXIX, 477; Lewis and Doisy, Ibid., 1918, XXXVI, i; Lewis, 
Dunn, and Doisy, Ibid., 9; Gudzent, Maase and Zondek, Ztschr. f. klin. Med., 1918. 
LXXXVI, 34; Host, Jour. Biol. Chem., 1919, XXXVIII, 17. 



THE URINE 245 

The amount of exercise will also influence the output in the urine; while an 
intake of a large amount of water will increase the normal uric acid value. A 
certain relation between the amount of urea and of uric acid excreted seems 
to obtain. As a rule, it may be said that the nitrogen of the urea is to the 
nitrogen of the uric acid as 50 or 60 to i. It is probable that variations in 
this relation have at present much less clinical value than in times past. It 
does not seem to the writer consistent to assume a special " uric acid diathesis" 
if this normal relation be disturbed in the sense that the uric acid is increased. 
Pathologic Variations. 

A pathologic increase of uric acid is observed whenever we have increased 
protein catabolism. Thus, in fever, the output of uric acid runs parallel to 
that of urea. In cases associated with marked leucocy tosis a large increase in 
the uric acid output may be noted, which is referable to the constant leucoly- 
sis. This increase is especially marked in leukemia, an output of more than 1 2 
grams in 24 hours having been observed by Magnus Levy. In cases of pneu- 
monia, associated with a high leucocyte count, an increase in the uric acid out- 
put is seen, being especially marked after the crisis, but may even precede the 
crisis. 

Gout^ has so long been associated, in the minds of the profession, with 
increased uric acid formation that such a relationship is generally accepted. 
It is true that during the acute attack the blood may contain an increased 
amount of uric acid, but never in such a large amount as in cases of leukemia, 
for instance. The mere excess of uric acid in the blood can, therefore, not be 
the determining factor. As our knowledge of the true etiology of gout is so ob- 
scure the writer will not attempt a discussion of uric acid in such relations. 
According to Futcher the uric acid is below the normal standard preceding 
the acute attack, rises to much increased values during the attack, and again 
falls below the normal limit after the subsidence of the acute symptoms. In 
acute articular rheumatism an increased elimination is noted during the 
febrile period, a decrease being observed as convalescence approaches. In 
diabetes mellitus an increase or a decrease may be observed, the uric acid 
excretion varying inversely as that of the sugar, giving rise to the term dia- 
betes alternans. In cases showing much degeneration of hepatic tissue, as in 
cirrhosis and acute yellow atrophy, uric acid may be largely increased. 

A diminished excretion of uric acid is usually observed in the ordinary 
forms of anemia while in pernicious anemia an increase may be noted. 
Chronic interstitial nephritis, chronic lead-poisoning, purpura hemorrhagica, 
and some cases of epilepsy, are associated with a diminished output of uric 
acid. Large doses of quinin or of opium may diminish the uric acid, while 
salicylic acid, chinic acid, colchicin; urotropin, piperazin, nucleinic acid, etc., 
may increase the amount.^ 

The chemical properties of uric acid may be found in works on physio- 
logic chemistry. Qualitative tests are frequently desirable for the detection 

1 See Benedict, Jour. Lab. and Clin. Med., 1916, II, i; Yavein, Russk. Vrach, 1916, XV, 
1203; McClure and Pratt, Arch. Int. Med., 191 7, XX, 481. 

2 See Denis, Jour. Pharmacol, and Exper. Therap., 1915, VII, 601; Haskins, Arch. Int. 
Med., 1915, XVI, 1055; Ibid., 1916, XVII 405; Jour. Biol. Chem., 1016, XXVI, 205. 



246 DIAGNOSTIC METHODS 

of the nature of certain deposits, as, for instance, renal infarcts. These will 
be discussed in a later section in association with the description of the 
various crystalline forms which uric acid may assume in a urinary deposit. 
Quantitative Determination. 
Folin and Shaffer's Method. 

This modification^ of Hopkins' method depends upon the precipitation of 
uric acid, as ammonium urate, by addition of ammonium sulphate. The 
urate is decomposed with sulphuric acid and the liberated uric acid is deter- 
mined by titration with a standard solution of potassium permanganate. 
Technic. 

Two hundred c.c. of urine are treated with 50 c.c. of a reagent consisting of 
500 grams of ammonium sulphate, 5 grams of uranium acetate, and 60 c.c. of 
10 per cent, acetic acid, dissolved in 650 c.c. of water. The mixture is allowed 
to stand without stirring for about one-half hour. The precipitate of uranium 
phosphate has then settled and the clear supernatant Hquid is removed by 
siphonage or by decantation. One hundred and twenty-five c.c. of this clear 
fluid, representing 100 c.c. of urine, are measured into a beaker, 5 c.c. of strong 
ammonia are added, and the mixture set aside until the following day. The 
precipitate of ammonium urate, which is produced by alkalinizing the solu- 
tion saturated with ammonium sulphate, is then filtered off, and washed with 
10 per cent, ammonium sulphate solution until the filtrate is practically free 
from chlorids. The filter is removed from the funnel, opened, and the precipi- 
tate rinsed back into the beaker, enough water to make about 100 c.c. being 
used. The precipitate is now dissolved by adding 15 c.c. of concentrated sul- 
phuric acid. The solution is then titrated with a twentieth-normal solution 
of potassium permanganate, until the first pink coloration is observed extend- 
ing through the entire liquid after the addition of two drops of the perman- 
ganate solution. Each c.c. of potassium permanganate used corresponds to 
3.75 mg. of uric acid. Owing to the solubility of the ammonium urate, a cor- 
rection of 3 mg. per 100 c.c. of urine must be made. The corrected result 
gives the percentage of uric acid.^ 

A method of making a twentieth-normal potassium permanganate solution 
may be found in any work in general chemistry. The student is to be cau- 
tioned that a twentieth-normal solution of potassium permanganate, as used 
in this connection, has reference to one of such a concentration that one liter 
would contain 0.05 gram of available oxygen for oxidizing purposes. This 
solution is obtained by dissolving 1.581 grams of pure KMn04 in one liter of 
water. As this weighing may not be sufficient, owing to slight impmrities in 
the permanganate, it is wise to titrate this solution against a known twentieth- 
normal solution of oxalic acid. If the solutions correspond, i c.c. of each 
should be the equivalents. The titration of the oxalic solution is made by 
measuring out 10 c.c. of oxalic acid solution, diluting to approximately 100 c.c. 
with distilled water, and adding 15 c.c. of concentrated sulphuric acid. The 

1 Ztschr. f. physiol. Chem., 1901, XXXII, 552. See Morris, Jour. Biol. Chem., 1919, 
XXXVII, 231, for a new titration method involving the use of potassium permanganate. 

2 Skorczewski und Sohn (Wien. klin. Wchnschr., 191 1, XXIV, 1700) show that the urine 
of patients taking atophan may reduce the permanganate and, hence, introduce^an error. 



THE URINE 247 

permanganate solution is then added drop by drop until a faint permanent 
red color is observed throughout the liquid, which does not disappear on stir- 
ring but persists for at least 30 seconds. If these solutions do not correspond 
the permanganate solution must be diluted according to the formula pre- 
viously given under the determination of the chlorids in the urine. 

Salkowski-Ludwig Method. 

This method is not as convenient as that of Folin, but is inserted as it serves as 
a combined method of determining uric acid and the purin bases at the same time. 

The principle is as follows: Uric acid is precipitated, in the form of a 
double urate of silver and magnesium, by an ammoniacal silver solution in the 
presence of magnesium salts. The silver is removed by hydrogen sulphid and 
the uric acid precipitated by hydrochloric acid, after which it may be esti- 
mated by direct weighing or by determination of the nitrogen. 

Technic. 

Two hundred and forty c.c. of urine are treated with 60 c.c. of magnesium 
mixture, which is made up as follows: One hundred grams of crystallized 
magnesium chlorid and 200 grams of ammonium chlorid are dissolved in 
about 500 c.c. of water, ammonia is added until the mixture smells strongly of 
this substance and the whole is then made up to one liter. The above mixture 
is well stirred and immediately filtered through a dry filter-paper into a beaker. 
Two hundred and fifty c.c. of filtrate (representing 200 c.c. of urine) are then 
measured off and treated with 10 to 15 c.c. of an ammoiuacal silver nitrate 
solution (one liter of which contains 26 grams of AgNOs and enough ammonia 
to dissolve the precipitate formed). The precipitate should be of a flocculent 
gelatinous nature and of a yellowish color. If the precipitate be white, too 
much silver chlorid is present and more ammonia must be added. The clear 
solution above the precipitate should contain an excess of silver chlorid which 
may be shown by adding a little nitric acid to a few drops of the clear super- 
natant liquid. This mixture is then filtered, any particles adhering to the 
beaker being transferred to the filter by means of water and a rubber 
tipped glass rod. The residue on the filter is washed with distilled water 
until the wash fluid shows no trace of silver or of chlorids. The funnel is now 
placed in the neck of a liter flask, the tip of the filter-paper is perforated with 
a glass rod, and the precipitate washed into the flask and thoroughly mixed 
with the water. This solution is made faintly acid by the addition of two 
or three drops of hydrochloric acid. Three to four c.c. of 10 per cent, copper 
sulphate solution are then added and the mixture boiled, after which hydro- 
gen sulphid is passed through the hot mixture to precipitate the silver salts. 
This gas should be passed until the solution is saturated with it, after 
which the solution is boiled and filtered. The precipitate is washed with 
hot water and the filtrate, which must be clear and colorless, is evaporated to 
a small bulk (10 to 15 c.c). Ten to fifteen drops of hydrochloric acid are 
then added, the mixture stirred and allowed to stand for a few hours, prefer- 
ably over night. This addition of hydrochloric acid precipitates the uric acid 
and leaves the purin bases in solution. The crystals of uric acid are filtered 



240 DIAGNOSTIC METHODS 

off on a small weighed filter, are washed with water slightly acidified with 
HCl to such an extent that the total wash- water and filtrate should not be 
more than 50 to 60 c.c. The precipitate is washed with alcohol, carbon 
disulphid, and ether, and is then dried and weighed. The difference be- 
tween the original weight of the filter-paper and that of the paper and pre- 
cipitate is the amount of uric acid in the 200 c.c. of urine. Owing to the 
slight solubility of uric acid in acidulated water, a correction of 0.00048 
gram must he added for every 10 c.c. of the filtrate and wash- water. 

Instead of weighing the uric acid, the filter-paper and contents may be 
placed in a Kjeldahl flask and a nitrogen determination made as previously 
described. The nitrogen value multiplied by 3 will give the weight of uric 
acid in 200 c.c. of urine. 

The filtrate and wash- water from the uric acid precipitation contains the 
purin bases. This filtrate is alkalinized with ammonia and again precipitated 
with the ammoniacal silver solution. This precipitate is collected on a small 
filter, washed with water, dried and carefully incinerated. The ash is dis- 
solved in nitric acid and the silver chlorid estimated by titration with potas- 
sium sulphocyanate as described under the determination of chlorids. One 
c.c. of the potassium sulphocyanate solution is equivalent to 0.00734 gram of 
silver. One part of silver, in the form of the silver compounds of the purin 
bases, represents 0.277 gram of nitrogen, or 0.7381 gram of the purin bases. 
It is evident, therefore, that i c.c. of the potassium sulphocyanate solution 
will represent 0.002 gram of nitrogen and 0.00542 gram of purin bases accord- 
ing to the following proportion: 

I : 0.00734 : : 0.277 • ^ 
X = 0.002 
By multiplying the number of c.c. of potassium sulphocyanate solution used 
to precipitate the chlorids by 0.00542 we obtain the number of grams of purin 
bases in the 200 c.c. of urine originally used. It is wise in determining the 
amount of purin bases to start with a larger quantity of urine, as, for instance, 
600 to 700 c.c, as the amount present in 200 c.c. would be very small. 

This method is apt to give slightly high values for uric acid, as the purin 
bases may not be entirely soluble in the acid solution used to separate them 
from the uric acid. It requires much more equipment than is usually at the 
disposal of the practitioner so that it can hardly be recommended for routine 
use in general work. 

Method of Rudisch and Kleeberg. 

This method^ is quite as accurate as is the preceding and has the advan- 
tage that it can be carried out in from 20 to 30 minutes. The principle is as 
follows: The total purin bodies are precipitated by an excess of silver nitrate, 
and the excess of silver determined volumetrically^ by potassium iodid, using 
a mixture of nitrous and sulphuric acid with starch solution as an indicator. 
As the silver compounds of the purin bases are soluble in strong ammonia 
solution, it is possible to make a determination of the uric acid and then esti- 

1 Am. Jour. Med. Sci., 1904, CXXVIII, 899. 

2 See Kretschmer, Biochem. Ztschr., 1913, L, 223. 



THE URINE 249 

mate the purin bases by subtraction of this value from the total purin 
compounds. 

Technic. 

One hundred and ten c.c. of urine are treated with 55 c.c. of fiftieth-normal 
AgNOs solution and diluted with strong ammonia to 220 c.c. (The fiftieth- 
normal solution of silver nitrate is made by dissolving 3.3932 grams of AgNOs, 
which has been heated for 10 minutes with a small amount of water to i2o°C., 
in 500 c.c. of water, adding 75 c.c. of strong ammonia and 10 grams of ammo- 
nium chlorid and making the whole up to one liter.) The addition of the 
ammonia dissolves the purin bases, leaving the uric acid in the precipitate. 
The original mixture is now filtered in such a way that two 100 c.c. portions 
are obtained, each portion representing 50 c.c. of urine. With the first of 
these portions an approximate estimation of the uric acid is made, while with 
the second the accurate one is carried out. 

To one of these portions, fiftieth-normal potassium iodid solution is added 
from a buret, a few drops being removed from time to time and added to a 
solution of nitrous-sulphuric acid mixed with a little starch paste. (The 
fiftieth-normal potassium iodid solution shotild contain 3.32 grams of KI in 
one liter. The nitrous-sulphuric acid mixture is made by mixing 25 c.c. of 
H2SO4 with 75 c.c. of H2O and i c.c. of fuming HNO3.) The addition of the 
drops of the solution to the indicator is for the purpose of determining the 
point at which an excess of potassium iodid occurs. This will be shown by the 
appearance of a distinctly blue contact ring. When the solution shows such a 
reaction, on adding a few drops of the mixture to the indicator, the number of 
c.c. of KI is read off, and we are then prepared for the more accurate control. 
It is wise to keep the indicator cold by immersion in ice-water, as otherwise a 
violent reaction may occur. 

The second 100 c.c. portion is then carefully titrated with the fiftieth-nor- 
mal KI solution by running directly about i c.c. less of this solution into the 
urine than was required in the first titration. After this point is reached the 
KI solution should be added drop by drop and a test made after the addition 
of each five drops. In this way an accurate end-point may be reached. As 
each 100 c.c. portion represents 50 c.c. of urine it will contain 25 c.c. of fiftieth- 
normal KI used and the number of c.c. of silver nitrate which combined with 
the uric acid of the urine is obtained. One c.c. of fiftieth-normal AgNOs 
solution represents 0.00336 gram of uric acid. By multiplying this factor by 
the number of c.c. of silver nitrate used, we obtain the amount of uric acid in 
50 c.c. of urine. 

The second part of this method consists in the determination of the total 
purins. One hundred and ten c.c. of urine are treated with 55 c.c. of fiftieth- 
normal AgNOs solution and diluted with water to 220 c.c. The remainder 
of the process is exactly as outlined above. The number of c.c. of fiftieth- 
normal silver nitrate used will represent the values for the total purins. 

If the number of c.c. of silver nitrate solution used in the previous deter- 
mination of uric acid be subtracted from the number used in the determina- 
tion of total purin, the result will be the number of c.c. referable to the purin 



250 DIAGNOSTIC METHODS 

bases. One c.c. of fiftieth-normal AgNOs solution represents 0.00152 gram of 
purin bases calculated as xanthin. By multiplying this factor by the number 
of c.c. of AgNOs used we obtain the amount of xanthin in 50 c.c. of urine. 

The writer has used this method frequently and has found its results very 
similar to those of the Salkowski-Ludwig method, although the amount of 
uric acid is somewhat less in this method than in the latter one. 

It is very simple, is quick and accurate and may, therefore, be recom- 
mended for general work. A somewhat similar method is advocated by 
Bartley.^ The writer finds this latter method fairly reliable and simple. 

Folin and Wu's Colorimetric Method. 

This method^ combines the use of some of the reagents used in other 
tests by Folin and his associates together with the introduction of a new 
permanent standard uric acid solution for comparison in the color tests. 

Solutions Necessary. 

1. Standard uric acid solution. Before starting to prepare the uric 
acid solution, a 20 per cent, filtered solution of sodium sulphite should be 
available. Dissolve i gram of pure uric acid m 125 to 150 c.c. of 0.4 per 
cent, lithium carbonate solution and dilute to a volume of 500 c.c. Trans- 
fer 50 c.c, corresponding to 100 mg. of uric acid, to each of a series of volu- 
metric Uter flasks. Add about 300 c.c. of water and then add 500 c.c. of clear 
20 per cent, sodium sulphite solution, mix, dilute to volume, and mix thor- 
oughly. Fill a series of 200 c.c. bottles with this solution and stopper very 
tightly in order to reduce the absorption of oxygen from the air. 

2. A 10 per cent, sodium sulphite solution, kept like the uric acid solution, 
in small tightly stoppered bottles. 

3. A 5 per cent, solution of sodium cyanide. 

4. A solution containing 5 per cent, of silver lactate and 5 per cent, of 
lactic acid. 

5. The uric acid reagent of Folin and Denis.^ This is prepared by boil- 
ing 100 grams of sodium tungstate* with 80 c.c. of "syrupy" phosphoric acid 
(85 per cent.) and 700 c.c. of water for not less than 2 hours (using a reflux 
condenser) and then diluting to i liter. When this reagent reacts with 
uric acid, a blue solution is formed, which permits of clear-cut color com- 
parisons. 

Technic. 

Transfer from i to 3 c.c. of urine to a 15 c.c. centrifuge tube and mix 

with enough water to make a volume of about 6 c.c. Add 5 c.c. of the acid 

silver lactate solution and stir with a fine glass rod (diameter i to 2 mm.). 

Rinse off the rod with a few drops of water and centrifuge. If enough silver 

1 Medical Chemistry, Philadelphia, 1904. 

2 Jour. Biol. Chem., 1919, XXXVIII, 459. 

3 See Folin and Macallum, Ibid., 191 2, XIII, 563; Folin and Denis, Ibid., 1913, XIV, 
95; Autenrieth and Funk, Munch. Med. Wchnschr., 1914, LXI, 457; Host, Ztschr. f. klin. 
Med., 1914, LXXXI, 113; Benedict and Hitchcock, Jour. Biol. Chem., 1915, XX, 619. 

* Egerer and Ford (Proc. Soc. Exp. Biol. Med., 1918, XVI, 10) call attention to the 
frequent necessity of purifying the tungstate before use. 



THE URINE 



251 



solution has been added, the precipitate settles very quickly. Add a drop 
of silver lactate solution, to insure an excess of this reagent; if a precipitate 
forms, add more (2 c.c.) and centrifuge again. Pour off the 
clear supernatant liquid as completely as possible. 

To the precipitate in the centrifuge tube add, from a 
buret, 4 c.c. of 5 per cent, sodium cyanide solution and 
stir until a perfectly clear solution is obtained. Pour the 
contents of the tube into a 100 c.c. volumetric flask and 
rinse the tube and stirring rod, using for this purpose about 
15 to 25 c.c of water. Add 5 c.c. of 10 per cent, sodium laio 
sulphite solution (to balance that in the standard uric acid 
solution). Dilute to a volume of about 50 c.c. 

Transfer to another 100 c.c. volumetric flask < c.c. of 



11,8 
11.6 
11,4 
11.24 

it.o 

10,8 
10.6 
10.4 
10.2 
10,0 



urino 
.0.175 
.0,178 
.0.181 
0.184 
0,l8t 
0.190 
0.193 
0.1 9S 
0.199 
0.202 
0.205 
0.209 
0.211 
0.215 
0.219 
0.221 
0.225 
0.228> 
0.231 
0,235 
0.238 
0.^^42 
0.245 
0,249 
0.252 
0.26 
0.28 
0.3 
0.33 
0,35 

0,41 
0,44 
0,47 
0,5 
0.55 
0,6 
0.653 
0.71 
,76 
0.8 
0.94 



the standard uric acid sulphite solution, containing 0.5 mg. 
uric acid; add 4 c.c. of the cyanide solution and dilute to 
about 50 c.c. Then add 20 c.c. of saturated sodium car- 
bonate solution to each -flask, mix, and finally add with 
shaking 2 c.c. of the uric acid reagent. Let stand for 3 to 
5 minutes, fill to the mark, mix, and make the color com- 
parison in the usual manner. Artificial light (with "day- 
lite" glass) is better than day light for this comparison. 

To determine the amount of uric acid in milligrams in 
the volume of urine taken, with the standard set at 20 mm., 
divide 10 by the reading of the unknown. 

Ruhemann's Method. 

This method is a very convenient clinical one, although its 
results are by no means as accurate as those of the preceding 
methods.^ What the general practitioner desires, as a rule, 
is to know whether the uric acid is increased or diminished 
and does not care as to the absolute value. Such results^ 
giving the total purins, may be obtained for clinical purposes 
by this method. 

It consists in the use of a specially graduated tube, the 
uricometer, in which are placed the reagents and the urine to 
be tested (see cut). The calibrations of the tube are such as 
to represent directly the amount of uric acid in parts per 
1000. The principle of the method is the decolorization of 
an iodin solution by the uric acid of the urine, and the meas- 
urement of the amount of urine which must be added to a 
definite amount of iodin solution to effect this decolorization. 

Technic. 
Carbon disulphid is placed in the tube up to the mark S, in such a way 
that the lower meniscus of this reagent rests upon the mark. A solution 
of iodin in potassium iodid is then added so that the upper portion of the 



Fig. 79. — 
Ruhemann's 
uricometer. 



See Bradley and Bunta, Jour. Am. Med. Assn., 19 13, LX, 44. 



252 DIAGNOSTIC METHODS 

meniscus coincides with the mark J. This iodin solution has the following 
composition. 

Iodin, 0.50 gram 

Potassium iodid, 1.25 grams 

Absolute alcohol, 7.50 grams 

Glycerin, 5.00 grams 

Distilled water, q.s., 100.00 grams 

The urine is added slowly by means of a pipet until the lowest calibration 
is reached. The glass stopper is inserted and the contents of the tube mixed 
by repeated inversion for about 15 seconds. The carbon disulphid absorbs 
the iodin, taking on a distinct purple coloration. If this amount of urine 
does not completely decolorize the iodin, shown by the porcelain-like 
color of the carbon disulphid solution, more urine is added and the tube 
again inverted for 15 seconds. This process is continued until repeated 
shaking of the tube causes the carbon disulphid to assume a pale pink color. 
The reaction is practically ended at this point, as by a little more shaking of 
the contents the indicator will assume the characteristic porcelain-white ap- 
pearance. This process requires from 6 to 15 minutes. The amount of 
uric acid is then read off directly from the tube in parts per liter. 

Should the urine contain less uric acid than can be read off from the cali- 
brations, a second test is made adding the iodin solution to the mark midway 
between S and J, the amount indicated on the tube being of course divided by 
2. Conversely, should the urine contain more uric acid than is represented 
by the lower calibration, one adds the iodin solution to the point above J and 
multiplies his reading by 1.5, or adds the iodin solution to the second mark 
above J and multiplies the reading by 2. 

With this method the urine must be acid in reaction. If the urine con- 
tains a sediment of the urates, it should be thoroughly shaken before being 
added, so that the urates may be in suspension. Any free uric acid which 
may have separated in the sediment is not determined in this method. 
Strongly colored urines have no influence upon the decolorization. The 
presence of sugar does not interfere with the results, but if albumin be present 
in large amounts it should be removed by acidifying with dilute acetic acid, 
boiling, and filtering. The writer has used this method very frequently and 
finds it very useful for rough estimation of the uric acid outputs. The puri- 
nometer, introduced by Hall,^ seems to be a much more reliable and useful 
instrument, as it employs the reagents required by the Salkowski-Ludwig 
method. 

(e) Purin Bases. 

These bodies have been called purin bases, alloxur bases, xanthin bases, 
and nuclein bases. They are of extreme importance from the standpoint of 
physiologic chemistry, but are, clinically, of less value than is uric acid. The 
following bodies have been isolated from the urine in various conditions: 
adenin, guanin, epiguanin, carnin, episarkin, xanthin, hypoxanthin, heter- 

1 The Purin Bodies, Phila., 1904. 



THE URINE 253 

oxanthin, paraxanthin, and methylxanthin. Certain methylated xanthin 
compounds are found in tea and coffee and are, therefore, introduced into the 
system as caffein, theobromin, and theophylHn, being excreted either as xan- 
thin or hypoxanthin. Xanthin (C5H4N4O2) is 2, 6, dioxypurin. 

These nuclein bases of the urine arise either from the nuclein of the food or 
from the increased nuclein metabolism of the system. Very little is known at 
the present time regarding the absolute variations in the excretion of these 
purin bases in the urine. Salkowski finds an excretion ranging between 
0.0286 and 0.0561 gram (calculated as xanthin), while Camerer regards an 
average output as 0.087 gram in 24 hours. It is interesting to note that a 
vegetable diet appears to increase this output more than a meat diet, 0.044 
gram being excreted, according to Camerer, on a meat diet and o.iii gram on 
a vegetable regime. This finding is the reverse of that for uric acid. As a 
general rule, it may be said that the output of purin bases is from 8 to 10 per 
cent, of that of uric acid, varying from 16 to 60 mg. per diem. It is evident 
that foods containing these substances should be absolutely interdicted in 
conditions which may be traceable to disturbances in the nuclein metabolism. 

An increase in the amount of uric acid is usually associated with an in- 
crease of the xanthin bases, but at times no such relations obtain, a decrease 
in these bodies being observed. In leukemia an excretion of 0.321 gram has 
been reported by Magnus-Levy.^ In certain cases of tuberculosis, nephritis, 
epilepsy, migraine, and pneumonia the output may be much increased. 
Edsall finds the urinary output increased as a result of X-ray treatment. 
Xanthin is occasionally found as a constituent of the urinary sediment and 
may form calculi. ^ 

(/) Creatinin (C4H7N3O). 
The older ideas regarding the excretion of this nitrogenous body have 
suffered a severe shock from the work principally of Folin, Shaffer, Hoogen- 
huyze and Verploegh, Mellanby, and Klercker. Acid urines were supposed to 
contain creatinin and little or no creatin; while alkaline urines were thought to 
show creatin instead of creatinin. It has been prOven, however, that "nor- 
mal fresh urine, whether acid or alkaUne, contains creatinin, and if the normal 
subject has not taken creatin in his food during the preceding days his urine 
will not contain creatin, whatever its reaction. There is no normal excretion 
of endogenous creatin,^ as this, when ingested, is largely retained in the body 
unless the food contains a large amount of protein." Thus Folin has shown 
that the excretion of creatinin on a diet yielding a urinary nitrogen value of 16 
grams, was 1.55 grams or 3.6 per cent, of total nitrogen; while on a nitrogen- 
free diet with a urinary nitrogen of 3.6 grams, the creatinin output was 0.6 
gram or 17.2 per cent, of total N. He showed, further, that while the actual 
amount excreted varied with different individuals, yet for the same person the 

^ Loc. cit. 

2 See Hefter, Deutsch. Arch. f. klin. Med., 1913, CIX, 322; also, Koch, Jour. Biol. Chem., 
1913, Xy, 43- 

^ This statement does not apply to growing children who show a normal excretion of a 
relatively large amount of creatin. See Mendel and Rose, Jour. Biol. Chem., i9ii,X, 213, 
255 and 265; also Folin and Denis, Ibid., 1912, XI, 253. 



2 54 DIAGNOSTIC METHODS 

output was practically constant, under the same conditions of health and mus- 
cular activity. He beUeves, therefore, that creatinin is by far the most re- 
liable index as to the amount of a certain kind of protein metabolism occurring 
daily in any given individual. He bases his conclusions upon the facts that 
" the absolute quantity of creatinin ehminated in the urine on a meat-free diet 
is a constant quantity, differing for different individuals, but wholly independ- 
ent of quantitative changes in the total amount of nitrogen eliminated." 
Shaffer^ believes that creatinin is not an index of the total endogenous protein 
catabolism, as patients in whom the endogenous catabolism is much in- 
creased may excrete very little creatinin. Folin finds that the chief factor 
determining the amount of creatinin eliminated on any special diet is the 
weight of the patient. Fat or corpulent persons yield less creatinin per unit 
of bod3^-weight than do lean ones. It is, therefore, necessary in metabolic 
work to consider not only the body-weight, but, also, the excess of fat in 
increasing the weight. 

According to Shaffer, the normal excretion of creatinin varies between 7 
and II mg. of creatinin- nitrogen per kilo of body- weight. In pathologic sub- 
jects it is low, varying from the normal to 2 mg. per kilo of body-weight in 24 
hours. He calls the creatinin-nitrogen excretion per kilo of weight the 
''creatinin coefficient." He thinks that creatinin is an index of some special 
process of normal metabolism taking place largely, if not wholly, in the mus- 
cles. Upon the intensity of this process appears to depend the muscular 
efficiency of the individual. In acute febrile conditions, in which an in- 
creased destruction of muscle tissue occurs, an increase is seen in the creatinin 
output during the active febrile period, while in the period of convalescence 
a diminished excretion will be observed. This excretion in fever does not 
run parallel to the muscular efficiency of the individual (Shaffer). Simon 
has shown that a diminished excretion occurs in anemia, marasmus, myositis 
ossificans, chlorosis, phthisis, chronic parenchymatous nephritis, progressive 
muscular atrophy, and pseudohypertrophic paralysis. 

According to Shaffer, creatin may be excreted by subjects of acute fevers, 
in the acute stages of exophthalmic goiter, in other conditions in which there 
is a rapid loss of muscle protein, and by women during the postpartum reso- 
lution of the uterus. Krause- has reported the constant presence of creatin 
in diabetes mellitus referable, probably, to a deprivation of carbohydrates. 
Although administration of carbohydrate usually diminishes the creatin ex- 
cretion, Folin's theory would indicate that an increased excretion of creatin 
may follow a carbohydrate-rich diet, if the state of nutrition of the body is 
high, as a greater fraction of the creatin of the food would be eliminated. 
The study of the excretion of creatinin and creatin in various conditions is 
fast becoming of the greatest importance.^ 

1 Am. Jour. Physiol., 1908, XXIII, i. 

2 Quart. Jour. Exper. Physiol., 1910, III, 289. 

3 See Mendel^ Science, 1909, XXIX, 584; Ditman and Welker, New York Med. Jour., 
1909, LXXXIX, 1000, 1046 and 1091; Ellis, Jour. Am. Med. Assn., 1911, LVI, 1870; 
Skutetzky, Deutsch. Arch. f. klin. Med., 191 1, CIII, 423; McCrudden, Jour. Exper. Med., 

1912, XV, 457; Sedgwick, Am. Jour. Dis. Child., 1912, III, 209; Kraus, Arch. Int. Med., 

1913, XI, 613; Scaffidi, Internat. Beitr. z. Ernahrungsstor., 1913, IV, 401, Orioli, Ibid., 421; 



THE URINE 



255 



Qualitative Tests for Creatinin. 

Creatinin has the formula, C4H7N3O, with the graphic structure 



HN 



HN = C 



H3C 




O 



CHs 



The chemical reactions which serve for the detection of creatinin depend 
upon the formation of different colored compounds. It forms a distinct crys- 
talline compound with zinc chlorid, which may be used in the quantitative 
estimation by Salkowski's method. For this process see works on physio- 
logic chemistry. A point to be remembered regarding creatinin is that it re- 
duces copper solutions and may be mistaken for sugar unless care be exercised. 

WeyPs Test. 
To the urine to be tested are added a few drops of a very dilute aqueous so- 
lution of freshly dissolved sodium nitroprussid and a few drops of dilute sodium 
hydrate solution. In the presence of creatinin a ruby-red color appears which 
changes, after a short time, to an intense yellow. If this solution be heated 
with a little glacial acetic acid the color will change to green and finally blue. 
Acetone gives a similar reaction, but on the addition of acetic acid changes to 
a purplish-red instead of green. If the urine be heated previous to the appli- 
cation of this test, the acetone may be driven off. This test for creatinin is 
sensitive to 6 parts in 10,000. 



Mellanby, Proc. Roy. Soc, 1913, LXXXVI, 88; Myers and Volovic, Jour. Biol. Chem., 
1913, XIV, 489; Myers and Fine, Ibid., 1913, XV, 283 and 305; Ibid., 1Q13, XVI, 169; 
Thomas and Goerne, Ztschr. f. physiol. Chem., 1914, XCII, 163; Lampert, Ztschr. f. klin. 
Med., 1914, LXXX, 498; FoHn and Morris, Jour. Biol. Chem., 1914, XVII, 469; Folin and 
Denis, Ibid., 493; Benedict, Ibid., 1914, XVIII, 183 and 191; Benedict and Osterberg, Ibid., 
195; Shaffer, Ibid., 525; Tracy and Clark, Ibid., 1914, XIX, 115; Palmer, Means and 
Gamble, Ibid., 239; Ringer and Raiziss, Ibid., 487; Hull, Jour. Am. Chem. Soc, 1914, 
XXXVI, 2146; Morris, Jour. Biol. Chem., 1915, XXI, 201; Myers and Fine, Ibid., 377, 383 
and 389; Janney and Blatherwick, Ibid., 567; Myers and Fine, Ibid., 583; Taylor, Ibid., 
663; Baumann and Marker, Ibid., 1915, XXII, 49; MacAdam, Biochem. Jour., 1915, IX, 
229; Practitioner, 1914, XCII, 540; Hutchison, Indian Jour. Med. Research, 1915, II, 814; 
van Hoogenhuyze, Nederl. Tijdschr. v. Geneesk., 1915, I, 1786; Detweiler and Grifl&th, 
New York Med. Jour., 1915, CII, 798; Morse, Jour. A. M. A., 1915, LXV, 1613; Tsuji, 
Biochem. Jour., 1915, IX, 449; Baumann and Marker, Jour. Biol. Chem., 1915, XXII, 
49; Rose, Ibid., 1916, XXVI, 331; Rose, Dimmitt and "Cheatham, Ibid., 339; Rose and 
Dimmitt, Ibid., 345; Denis, Ibid., 379; Underbill, Ibid., 1916, XXVII, 127 and 141; 
Underbill and Baumann, Ibid., 147, 151 and 169; Underbill and Bogert, Ibid., 161; 
Burns and Orr, Biochem. Jour., 1916, X, 495; Powis and Raper, Ibid., 363; Cutter and 
Morse, Am. Jour. Dis. Child., 1916^ XI, 326 and 331; Veeder and Johnston, Ibid., 1916, 
XII, 136; Lyman and Trimby, Jour. Biol. Chem., 191 7, XXIX, i; Denis, Ibid., 447; 
Ibid., 1917, XXX, 47; Denis and Kramer, Ibid., 189; Baumann and Hines, Ibid., 1917, 
XXXI, 549; Denis and Minot, Ibid., 561; Rose, Ibid., 191 7, XXXII, i; Hunter and 
Campbell, Ibid., 1918, XXXIV, 5; Rose, Dimmitt and Bartlett, Ibid., 601; Baumann 
and Hines, Ibid., 1918, XXXV, 75; Baumann and Ingvaldsen, Ibid., 277; Steenbock and 
Gross, Ibid., 1918, XXXVI, 265; McClure, Arch. Int. Med., 1918, XXII, 719; Denis 
and Minot, Jour. Biol. Chem., 1919, XXXVII, 245; Gamble and Goldschmidt, Ibid., 
XL, 199 and 215. 



256 ' DIAGNOSTIC METHODS 

Jaffe's Test. 

To the urine to be tested are added a few drops of a saturated solution of 
picric acid and a few drops of dilute sodium hydrate solution. If creatinin be 
present a red color appears immediately, which increases in intensity and 
remains permanent for a long time. If glacial acetic acid be added the color 
becomes yellow. Acetone gives a reddish-yellow color of less intensity than 
that produced by creatinin. Glucose, if present, may give a red color if the 
mixture be warmed. This test indicates one part of creatinin in 5,000. 

Quantitative Determination. 
Folin's Older Method. 

The principle upon which this determination^ is based is the comparison of 
the color produced by Jaffe's reaction with that of a standard solution of 
potassium bichromate. A high-grade colorimeter is necessary for this com- 
parison. Folin recommends the use of the Duboscq instrument, while the 
writer finds one made by Sargent & Co., of Chicago, very satisfactory. ^ This 
latter has the advantage of being much less expensive. 

The reagents necessary are (i) a half-normal potassium bichromate solu- 
tion containing 24.55 grams per liter, (2) a saturated picric acid solution contain- 
ing about 1 2 grams per liter, ^ and (3) a 10 per cent, solution of sodium hydrate. 

Technic. 

Ten c.c. of urine are measured into a 500 c.c. volumetric flask, 15 c.c. of 
the picric acid solution and 5 c.c. of the sodium hydrate solution are then 
added, and the mixture allowed to stand for five or six minutes. This interval is 
used to pour a httle of the standard bichromate solution into each of the two 
cylinders of the colorimeter. The depth of the solution in one of the cylinders 

^ Am. Jour. Physiol., 1905, XIII, 45. 

2 See Kober, Jour. Biol. Chem., 1917, XXIX, 155; Bock and Benedict, Ibid., 1918, 
XXXV, 227. 

^ As it had been shown by McCrudden and Sargent (Jour. Biol. Chem., 1916, XXIV, 
423 and 527) as well as by Hunter and Campbell (Ibid., 191 7, XXVIII, 335) that serious 
errors and discrepancies were obtained by the use of Folin's method; Folin and Doisy 
(Ibid., 349) found that the trouble was due to impurities in the picric acid as obtainable on 
the market. For this reason the picric acid, used in determinations of creatinin, should 
either be known to be pure when purchased or should be purified by the following method. 
Transfer about 600 grams of wet picric acid, or about a pound of dry picric acid, to a large 
beaker (capacity not less than 4 liters). Pour on boiling water until the beaker is nearly 
full and add 200 c.c. of saturated (50 per cent.) sodium hydrate solution. Stir, and if 
necessary heat again until all the picric acid has dissolved, yielding a deep red picrate 
solution. To the hot solution add, rather slowly, with stirring, 200 grams of sodium 
chlorid. Cool in running water to about 3o°C., with occasional stirring. Filter on a large 
Buchner funnel and wash a few times with 5 per cent, sodium chlorid solution. Transfer 
the picrate to a large beaker, fill with boiling water, and when the picrate is dissolved add, 
with stirring, first 50 c.c. of 10 per cent, sodium hydrate solution, and then 100 grams of 
sodium chlorid. Cool to 3o°C., with stirring, filter, and wash with sodium chloride solu- 
tion, as before. Repeat the solution and precipitation of the sodium picrate twice more, 
but for the last washing of the last precipitated picrate use distilled water instead of sodium 
chlorid solution. Dissolve the purified picrate in the same large beaker, with boiling dis- 
tilled water, and filter hot on a large folded filter, collecting the filtrate in a large flask. 
To the hot filtrate add 100 c.c. of concentrated sulphuric acid, previously diluted with 
about two volumes of water. The liberated picric acid begins to come out at once. 
Put a beaker over the mouth of the flask and cool under running tap water to about 30° 
C. Filter with suction and wash free from sulphates with distilled water. 



THE URINE 



257 



is then accurately adjusted to the 8 mm. mark. With the solution in the other 
cylinder a few preliminary colorimetric readings are made simply for the sake 
of insuring greater accuracy in the subsequent readings of the unknown solu- 
tion. The two bichromate solutions must, of course, be equal in color, and in 
taking their readings no two should 
differ more than o.i mrn. or 0.2 mm. 
from the true value (8 mm.), leaving 
out of consideration the very first 
reading made, which is sometimes 
less accurate. Four or more readings 
should be made in each case, and an 
average taken of all but the first. 
After a while one becomes sure of the 
true point, and can take the average 
of the first two readings. 

At the end of five minutes the 
contents in the 500 c.c. flask are di- 
luted up to the 500 c.c. mark. The 
bichromate solution is thoroughly 
rinsed out of one of the cylinders by 
means of the unknown solution and 
several colorimetric readings are then 
made at once. The calculation of the 
results is very simple. It is based on 
the experimentally determined fact 
that 10 mg. of perfectly pure creati- 
nin give, under the conditions of the 
determination, 500 c.c. of a solution, 
8.1 mm. of which have exactly the 
same colorimetric value as 8 mm. of a 
half -normal bichromate solution. ^ 

.If, for example, it is found that it takes 9.5 mm. of the unknown urine- 
picrate solution to equal the 8 mm. of the bichromate, then the 10 c.c. of urine 
contain 

8-1 o , . . . 

10 X — = 8.4-f mg. of creatmm. 
9-5 ^ ' 6 




Fig. 80.— Sargent's colorimeter. (Courtesy 
of E. H. Sargent & Co.) 



If the 10 c.c. of urine used in the test are found to contain more than 15 
mg. or less than 5 mg. of creatinin, the determination should be repeated with 
a correspondingly different amount of urine, because outside of these limits 
the determination is much less accurate. The color of the urine does not 
materially affect the results on account of the great dilution. Sugar and albu- 



^ This relationship applies to the Duboscq instrument, which Folin advises. If one uses 
the Sargent or other colorimeter, he must, of course, make his adjustment according to his 
special instrument. It matters little what point is used for the standard solution, provid- 
ing one has tested his bichromate solution against solutions of creatinin treated as above 
and has obtained his relative values. 



258 DIAGNOSTIC METHODS 

min do not interfere with the determination, while acetone and diacetic acid 
do unless one allows a few minutes before reading.^ 

Folin's Newer Method. 

In order to enlarge the scope of the colorimetric method for the determina- 
tion of creatinin and creatin, Folin advises the replacing of the bichromate 
solution with a standard creatinin solution for the color comparison. While 
creatinin may be obtained on the market it is usually far from pure, so that 
one should prepare his own creatinin according to the method of Folin^ 
or of Benedict.^ 

The method is as follows: One c.c. of a standard solution of creatinin (i 
gram per liter, hence i c.c. containing i mg.)^ is measured into a 100 c.c. volu- 
metric flask and i c.c. of the urine into a similar flask by means of the accurate 
Ostwald pipets (care being taken to allow the pipet to drain against the neck 
of the flask for a few seconds and then to blow out the pipet clean). Twenty 
c.c. of saturated picric acid solution (measured with a cylinder) are added to 
each flask and then 1.5 c.c. of 10 per cent, solution of sodium hydrate are in- 
troduced from a buret. At the end of 10 minutes the flasks are filled up to the 
mark with tap water, are mixed thoroughly and the relative color of the two 
solutions determined in the colorimeter. It makes Uttle difference whether 
the standard solution is set at 10, 15 or 20 mm., as the comparisons are equally 
exact at any one of these points. This standard reading is divided by the read- 
ing of the unknown to obtain the milligrams of creatinin present in the volume 
of urine (i c.c.) taken. If the urine reads less than two- thirds or more than 
one and one-haK that of the standard the determination should be repeated 
with more or less urine as the case may be. 

Creatin. 

The determination of creatin, as well as of creatinin, is of much importance 
in various conditions. Many methods have been given for such work but the 
following seems to offer the best results with the simplest conditions. 

Folin's Method.^ 

Enough urine to give 0.7 to 1.5 mg. of creatinin is measured into a weighed 
Erlenmeyer Jena flask, (capacity 200 c.c). Twenty c.c. of a saturated picric 
solution, about 130 c.c. of water and a few very small beads to promote even 
boihng are added and the mixture is gently boiled, preferably over a micro- 
burner, for about i hour. At the end of this time increase the heat and boil 
down the solution to about 20 c.c. Transfer the flask to the scales 
and add enough water to make the total solution equal to 20 to 25 grams. 
Cool the solution in running water, add 1.5 c.c. of 10 per cent, sodium hydrate 

^ See Greenwald, Jour. Biol. Chem., 1913, XIV, 87; also, Thompson, Wallace and Clot- 
worthy, Biochem. Jour., 1913, VII, 445. See Dehn, Jour. Am. Chem. Soc, 1917, XXXIX, 
1392 for possible errors in this test; also, Allen and Davisson, Jour. Biol. Chem., 1919, 
XL, 183. 

2 Jour. Biol. Chem., 1914, XVII, 463 and 469. 

3 Jour. Biol. Chem., 1914, XVIII, 183. 

* Instead of the creatinin itself, one may use the creatinin-zinc chlorid dissolving 
1. 6 1 06 grams in a liter of N/io hydrochloric acid. This salt is more easily prepared ii^ a 
pure state than is the creatinin. 

^ Jour. Biol. Chem., 1914, XVII, 469. See, also, Benedict, Ibid., 1914, XVIII, 191. 



THE URINE 259 

solution and determine the total creatinin as in the above method, using i mg. 
of pure creatinin solution as a standard. 

This method gives both the preformed creatinin and that derived from the 

creatin. Subtract the preformed (as determined by the direct method) from 

the total creatinin and obtain the creatinin derived from creatin. Multiply 

this latter figure by 1.16 to determine the actual amount of creatin as such. 

(g) Undetermined Nitrogen. 

By the undetermined nitrogen of the urine is meant the nitrogen remaining 
after that attributable to urea, uric acid, xanthin bases, ammonia, and creati- 
nin has been subtracted from the total nitrogenous output. This factor is 
made up of many substances present in variable amount and determined with 
more or less difficulty.^ 

As the variations in the other nitrogenous constituents have been shown to 
depend to a great extent upon the nature of the diet, so we would expect the 
values for the undetermined nitrogen to show similar fluctuations. Folin 
finds on a diet yielding 16 grams of total nitrogen in the urine an undetermined 
nitrogen output of 0.6 gram or 3.75 per cent, of total nitrogen; while on a 
nitrogen-free diet with a urinary nitrogen value of 3.6 grams the undetermined 
nitrogen is 0.27 gram or 7.3 per cent. He is led, therefore, to make the follow- 
ing generahzation: ''The absolute quantity of undetermined nitrogen de- 
creases under the influence of the starch and cream diet, but in per cent, of the 
total nitrogen there is always an increase." The separation of the various 
factors included in this undetermined nitrogen has clinically little value at 
the present time. 

(i) Amino Acids. 

Theoretically, we should find in the urine, whenever hepatic metabohsm 
is disturbed, both mono- and di-amino acids, as these substances are normal 
products of protein hydrolysis in the intestine and are directly converted into 
urea in the normal liver. It has long been known that acute yellow atrophy 
and phosphorus poisoning were associated with a diminished urea output and 
the presence of the two mono-amino acids, leucin and ty rosin, in the urine. 
Recent work^ with improved methods has shown that the urine contains these 
and other amino acids in any condition in which hepatic activity is impaired, 
so that our old-time diagnostic point of acute yellow atrophy must fall by the 
wayside. Although these substances are found in much larger quantities 
under pathologic conditions, traces of them are present in normal urine, 

^ Webster, Brit. Med. Jour., 1916, II, 845, has advanced a method for detection of small 
amounts of certain organic nitro compounds in the urine of TNT workers. See, also, 
Elvove, Jour. Indus, and Eng. Chem., 1919, XI, 860. 

2 See Signorelli, Biochem. Ztschr., 1912, XXXIX, 36; Ibid., 1912, XL VII, 482; Galam- 
bos and Tausz, Ztschr. f. klin. Med., 1913, LXXVII, 14; Damask, Ztschr. f. klin. Med., 

1913, LXXVII, S33', Hugounenq, Presse med., 1913, XXI, 825; Van Slyke and Meyer, Jour. 
Biol. Chem., 1913, XVI, 213; and Loffler, Ztschr. f. khn. Med., 1913, LXXVIII, 483; 
Benedict and Murlin, Jour. Biol. Chem., 1913, XVI, 385; Labbe and Bith, Rev. de. med., 

1914, XXXIV, 89; Udaondo and Casteigts, Sem. med., 1914, XXI, 741; Geake and Nieren- 
stein, Ztschr. f. physiol. Chem., 1914, XCII, 149; FoHn, Jour. Am. Med. Assn., 1914, 
LXIII, 823; Harding and MacLean, Jour. Biol. Chem., 1915, XX, 217; Van Slyke, Ibid., 
XXIII, 407; Bang, Biochem. Ztschr.. 1916, LXXII. loi; Harding and MacLean, Jour. 
Biol. Chem., 1916, XXIV, 503; Van Slyke, Arch. Int.' Med., 1917, XIX, 56; De Graaff 
and van der Zande, Pharm. Weekblad, 1916, LIII, 1378. 



26o DIAGNOSTIC METHODS 

especially when the nitrogen intake has been large. Thus von Noorden re- 
gards glycocoll (amino-acetic acid) as a normal urinary constituent, its excre- 
tion averaging i gram daily. These acids are also found in cases of gout, 
pneumonia, especially during the absorption of the exudate, in diabetes, and 
in leukemia.^ 

A large number of these mon-amino acids have been isolated from the 
direct products of protein hydrolysis, but the number found in the urine has 
not been as large owing to the uncertain methods of examination. The intro- 
duction by Fischer of the esterification method has added much to our know- 
ledge of these bodies. By the di-amino acids we mean the bodies lysin,^ argi- 
nin and histidin, which are collectively known as the hexone bases. 
Benedict and Murlin's Method. 

This method^ is not as accurate as the gasometric one of Van Slyke,'^ but is 
much more suitable for clinical work. 

Measure into a 500 c.c. Erlenmeyer flask 200 c.c. of a 24-hour specimen of 
urine, which has been diluted to 2,000 c.c. Add an equal quantity of a 10 per 
cent, solution of Merck's phosphotungstic acid in 2 per cent, hydrochloric acid. 
Let the mixture stand at least 3 hours, preferably over night. Pour off 250 c.c. 
of the clear supernatant fluid, add i c.c. of a 0.5 per cent, alcoholic solution of 
phenolphthalein and then barium hydrate in substance until the whole fluid 
turns decidedly pink (this latter substance should be added a little at a time to 
avoid an excess). Let this mixture stand for i hour. Now filter off two 100 
c.c. samples, each representing 50 c.c. of urine. Neutralize these specimens 
to litmus paper by means of N/5 hydrochloric acid and add at once, to each, 10 
to 20 c.c. of formalin which has been previously neutralized with N/io sodium 
hydrate solution. Titrate the mixture cautiously with N/io NaOH to a deep 
red color, i.e., until the drop of alkali produces no additional color. Make the 
duplicated etermination in the same way and correct the results by deduction 
of the amount of N/io NaOH necessary to produce the same depth of color in 
an equal quantity of C02-free water with the same quantity of neutral forma- 
lin added. Each c.c. of N/io NaOH used in the titration, corrected as above, 

^ Warfield (Jour. Am. Med. Assn., 1914, LXII, 436) has attempted to base a diagnostic 
test for pregnancy on the presence in the urine of dialyzable products reacting to ninhydrin. 
This test, however, has no value as the urine, normal or abnormal, always contains dialy- 
zable substances, especially amino acids and certain ammonium compounds, which under 
proper conditions mil react to ninhydrin. In this connection see Abderhalden's test in the 
section on Blood; also, Jamison, Jour. Am. Med. Assn., 1914, LXII, 1084; Fischer, Ibid., 950 
and 1575; Falls and Welker, Ibid., 1800; Berman, Am. Jour. Obs., 1914, LXX, 192; Holmes, 
Lancet- Clinic, 1914, CXI, 400. 

Malone (Jour. Am. Med. Assn., 1915, LXIV, 1651), using a modification of the technic of 
Kiutsi (Urine Diagnosis by Means of Filtration Process, Sapporo, Japan, 1914), removes the 
biuret-reacting urinary substances by means of the addition of 0.3 gram of kaolin to 15 c.c. 
of urine, filters this mixture and neutralizes the filtrate. This is then submitted to the usual 
Abderhalden technic, employing 10 c.c. of the biuret-free filtrate and 0.2 gram of dried 
placenta. This latter test may prove to be of some diagnostic value. See Cutler and 
Morse, Jour. A. M. A., 1916, LXVI, 559; Berkowitz, Arch. Int. Med., 1917, XIX, 397. 

2 See Ackermann and Kutscher (Ztschr. f. Biol., 191 1, LVII, 355) who report the pres- 
ence of lysin in a patient with cystinuria. Kocher (Jour. Biol. Chem., 191 5, XXII, 295) 
finds these diamino acids largely increased in malignant tissues. Edlbacher, Ztschr. f. 
physiol. chem., 1915, XCV. 81. 

3 Jour. Biol. Chem., 1913, XVI, 385. 

* Ibid., 121 and 125. See, also, Chodat and Krummer, Biochem. Ztschr., 1914, LXV, 
392; Harding and MacLean, Jour. Biol. Chem., 1915, XX, 217. 



THE URINE 261 

represents 0.0014 gram of nitrogen referable to the amino acids. Multiply 
the nitrogen value by 2 to obtain the percentage, and this latter amount by 20 
to derive the total amino-acid nitrogen in the 24-hour specimen. 

(2) Hippuric Acid (C9H9NO3). 
This acid is a normal constituent of urine and varies between o.i and 
I gram in 24 hours. It is derived to a large extent from foods containing 
benzoic acid, such as prunes, cranberries, green-gages, bilberries and many 
other fruits, and is also formed from the metabolism of tissue and food protein. 
This substance is directly synthesized in the system by the combination of 
benzoic acid with glycocoll, but the exact place of synthesis is uncertain. A 
pathologic increase in the excretion of hippuric acid has been observed in 
acute febrile diseases, marked intestinal putrefaction, hepatic disturbances, 
and in diabetes mellitus, while in cases of acute diffuse and chronic paren- 
chymatous nephritis as well as in amyloid kidneys hippuric acid is practi- 
cally absent from the urine. ^ 

(3) Oxyproteic and Alloxyproteic Acids. 

These acids have been isolated from the urine and seem to be constant 
constituents, derived from protein catabolism. Both of them contain sul- 
phur and have been credited with forming a large percentage of the neutral 
sulphur output as well as contributing 2 to 3 per cent, of the total N excretion. ^ 
Bondzynski believes the oxyproteic acid accountable for Ehrlich's diazo- 
reaction (page 352). Salkowski^ has shown that a certain amount (3 to 4 
per cent.) of the urinary nitrogen is precipi table by absolute alcohol and is 
non-dialyzable. This is the so-called " Colloidal N" and is largely referable to 
oxyproteic acid and N-containing carbohydrates. As this factor is increased 
in many cases of carcinoma up to 9 or 10 per cent, of total N, he believes 
this may be of value in diagnosis. However, numerous workers have demon- 
strated that this increase does not always obtain in cases of carcinoma and is 
found in pregnancy, diabetes, some hepatic disorders and in pulmonary tuber- 
culosis. Salmon and Saxl have outlined a method for the determination of 
the sulphur factor of this oxyproteic acid as related to total N, and believe 
that an increase in this factor is indicative of carcinoma.^ 

(4) Allantoin (C4H6N4O3). 
This substance is a product of oxidation of uric acid. It is somewhat 
variable in amount, usually being found only in traces, but after a large 

^ Dakin, Jour. Biol. Chem., 1910, VII, 103; Steenbock, Ibid., 1912, XI, 201; Folin and 
Flanders, Ibid., 257; Hryntschak, Biochem. Ztschr., 1912, XLIII, 315; Raiziss and Dubin, 
Jour. Biol. Chem., 1915, XX, 125; Lackner, Levinson and Morse, Biochem. Jour., 1918, 
XII, 184. 

2 See Browinski and Dombrowski, Ztschr. f. physiol. Chem., 1912, LXXVII, 92; 
Domansky, Monatsschr. f. Psych, u. Neurol., 1912, XXXI, 53; Erben, Prager med. Wchn- 
schr., 1912, XXXVII, 427. 

2 Berl. klin. Wchnschr., i905,XLII, 1581 and 1618; Ibid., 1910, XL VII, 533, 1746 and 
2297. 

^ Marriott and Wolf, Am. Jour. Med. Sc, 1907, CXXXIII, 404; Mancini, Deutsch. Arch, 
f. klin. Med., 1911, CIII, 288; Caforio, Berl. klin. Wchnschr., 1911, XL VIII, 1843; Kojo, 
Ztschr. f. physiol. Chem., 1911, LXXIII, 416; Salomon and Saxl, Wien. klin. Wchnschr., 
1911, XXIV, 449; Riforma Med., 1911, XXVII, 421; Deutsch. med. Wchnschr., 1912, 
XXXVIII, 58; Neuberg, Der Harn, 1911, 788; Semenow, Folia Urol., 1912, VII, 215; Berl. 



262 DIAGNOSTIC METHODS 

intake of meat especially rich in nuclein the amount may be quite percep- 
tible. It is found in fairly large quantities in the urine of the new-born, in 
the amniotic fluid, in ascitic fluid, in traces in the urine of pregnancy, and in 
leukemic blood. Pathologic variations have been little studied. 

The best method for the determination of this substance is that of 
Wiechowski.^ A point to be remembered in connection with allantoin is that 
it may be present in the urine in sufficient quantities to reduce copper solu- 
tions and must, therefore, not be mistaken for sugar. 

(2) Fatty Acids. 

Traces of volatile fatty acids are present in all normal urines. The most 
important of these are formic,^ acetic, propionic, and butyric acids. They are 
doubtless formed in the intestinal tract by bacterial action upon the carbo- 
hydrates of the food, and may, therefore, be to some extent an index of the 
degree of carbohydrate fermentation. Their normal amount does not much 
exceed o.oi gram in 24 hours, although BlumenthaP gives the figures under 
an ordinary diet as being equivalent to from 50 to 80 c.c. of tenth- normal sul- 
phuric acid (0.25 to 0.39 gram). 

These acids are increased in febrile conditions, the amount running 
parallel to the rise in temperature. This increase in the fatty acids of the 
urine is known as lipaciduria. According to Rosenfeld, this increase in febrile 
states is observed only in those cases in which absorption of decomposing al- 
buminous material occurs, as in all suppurative processes within the system. 
In the convalescent stage of pneumonia these acids may be excreted in in- 
creased amount, while a diminished output is usually observed preceding the 
crisis. In hyperacidity of the gastric contents the fatty acids of the urine are 
increased, while in hypoacidity they are diminished. In contradistinction to 
the increase of fatty acids in cases of fever associated with suppurative con- 
ditions, we find a diminution in the amount of fatty acid in scarlet-fever, 
erysipelas, measles, and diphtheria. In cases of acute rheumatism formic 
acid is said to be excreted in large amounts. In cyclic vomiting lactic acid^ 
may be increased. 

klin. Wchnschr., 1913, L, 1436; Greenwald, Arch. Int. Med., 1913, XII, 283; Pribram, 
Munch, med. Wchnschr., 1913, LX, 2047; Dozzi, Gazz. d. osp., 1913, XXXIV, 1007; 
Marenduzzo, Riforma Med., 1913, XXIX, 1149; Thar and Beneslawski, Biochem. Ztschr., 

1913, LII, 435; Lehmann, Deutsch. Arch. f. klin. Med., 1913, CXII, 376; Stadtmiiller and 
Rosenbloom, Arch. Int. Med., 1913, XII, 276; Sassa, Biochem. Ztschr., 1914, LXIV, 195; 
de Bloeme, Swart and Terwen, Miinch. med. Wchnschr., i9i4,LXI, 1718; Biochem. Ztschr., 

1914, LXV, 345; von Furth, Ibid., 1915, LXIX, 448; Goodridge and Kahn, Biochem. Bull., 

1915, IV, 118; Damask, Wien. klin Wchnschr., 1915, XXVIII, 499; Goodridge and Kahn, 
Biochem. Bull, 1915, IV, 118. 

^ Beitr. z. chem. Physiol, u. Path., 1908, XI, 129; Biochem. Ztschr., 1909, XIX, 378; 
Ibid., 1910, XXV, 431. See, also, Plimmer and Skelton, Biochem. Jour., 1914, VIII, 641; 
Givens, Jour. Biol. Chem., 1914, XVIII, 417; Pepper and Grier, Jour. Infect. Dis., 1916, 
XIX, 694; Harding and Young, Jour. Biol. Chem., 1919, XL, 227. 

2 See Dakin and Wakeman, Jour. Biol. Chem., 191 1, IX, 329; Steppuhn and Schellback, 
Ztschr. f. physiol. Chem., 191 2, LXXX, 274; Dakin, Janney and Wakeman; Jour. Biol. 
Chem., 1913, XIV, 341; and Strisower, Biochem. Ztschr., i9i3,LIV, 189, for a discussion of 
formic acid as an intermediary metabolic product of both carbohydrates and protein. 

3 Pathologie des Harnes, Berlin, 1903. 

* For methods of determination see von Furth and Charnass, Biochem. Ztschr., 1910, 
XXVI, 199; Mondschein, Ibid., 1912, XLII, 91 and 105; also, Ishihara, Ibid., i9i3,L, 468; 
Levene and Meyer (Jour. Biol. Chem., 1913, XV, 65) have shown that lactic acid is a prod- 



I 



THE URINE 263 

\ 
A simple method of determining the fatty acids of the urine is to acidify 
from 250 to 500 c.c. of urine with 50 to 75 c.c. of dilute sulphuric acid and 
distill. The distillate is then titrated with tenth-normal sodium hydrate 
solution using phenolphthalein as an indicator. The results are expressed 
in terms of the corresponding number of c.c. of tenth-normal acid. 

(3) Oxalic Acid. 

The amount of oxalic acid eHminated in the urine in 24 hours varies from 
10 to 20 milligrams. A portion of this excretion is undoubtedly derived from 
the diet, but some of it is produced in the metabolism of the tissues, especially 
of those containing nuclein substances. Although the carbohydrates do not 
take part in the production of oxalic acid normally, we find, in conditions asso- 
ciated with fermentation of the carbohydrates in the stomach, a large increase 
in the output of oxalic acid. As oxahc acid is more or less readily formed 
by oxidation of uric acid, passing through the intermediate stage of oxaluric 
acid, we can readily see that increase of nuclein metaboUsm may increase the 
output of this substance. Strangely enough, it has been found that the in- 
take either of pure nuclein or of nuclein-containing foods was not associated 
with a corresponding increase in the urinary oxahc acid, so that we must 
assume that a large portion of this intake escapes in the feces in the form 
of calcium oxalate. It has, moreover, been found that the administration of 
oxalates by the mouth is not associated with increased excretion either in the 
urine or feces, so that we must assume a decomposition of the oxalic acid into 
carbon dioxid and water somewhere in the system. This conversion appears 
to take place in the intestinal canal under the influence of bacterial action. 
Crystals of calcium oxalate are frequently found in the urine in cases showing 
a marked increase in the output of ethereal sulphates, so that we must either 
assume an increased formation from the carbohydrates of the food, an in- 
creased intake and absorption of oxalates of the food, or an increase in the 
nuclein metabohsm as a result of absorption of the toxic products of intestinal 
putrefaction.^ 

Among the foods which are known to contain relatively large amounts 
of oxalic acid we find spinach, rhubarb, tomatoes, carrots, celery, string-beans, 
green peas, potato, figs, plums, strawberries, pepper, cocoa, tea, and coffee. 
Those which contain very httle are meat, milk, eggs, butter, cereals, rice, 
asparagus, cucumbers, mushrooms, lettuce, cauliflower, cabbage, pears, 
peaches, grapes, and melons. 

The increased elimination of oxalic acid may or may not be associated 
with a deposition of crystals of calcium oxalate. An increased ehmination is 
observed in cases associated with irregular activity of the gastro-intestinal 
tract. These cases usually show many types of nervous disorder, especially 

uct of the intermediary metabolism of carbohydrates. See, also, Yoshikawa, Ztschr. f. 
physiol. Chem., 1913, LXXXVII, 382; von Fiirth, Biochem. Ztschr., 1914, XLIV, 131; Ibid., 
156; Wien. klin. Wchnschr., 1914, XXVII, 877; UnderMll and Steele, Am. Jour. Dis. 
Child., 1914, VIII, 127: Schneyer, Biochem. Ztschr., 1915, LXX, 294; Maver, Jour. 
Biol. Chem., 191 7, XXXII, 71. 

^ See Wegrzynowski, Ztschr. f. physiol. Chem., 1913, LXXXIII, 112; also, Aron and 
Franz, Monatsschr. f. Kinderh., 1914, XII, 645. 



264 DIAGNOSTIC METHODS 

neurasthenia, and are characterized by the large deposit of oxalate crystals in 
the urine. To this condition has been given the name oxaluria, which can 
hardly be dignified as a definite pathologic entity. No inference can actually 
be drawn regarding the degree of elimination of oxalic acid from the ap- 
pearance of a deposit in the urine, as it has been shown by Fiirbringer that 
the urine may contain a large amount of oxalic acid without a sediment of 
calcium oxalate crystals being formed.^ Such cases, however, should be 
watched more or less closely as separation of calcium oxalate may occur 
within the pelvis of the kidney and lead to the formation of a calculus. In 
cases of jaundice a marked oxaluria may be observed which is directly ref- 
erable to the associated cholemia. In occasional cases of diabetes mellitus 
the elimination of oxalic acid may be much increased. 

Quantitative Determination. 
Baldwin's Method. ^ 
Five hundred ex. of a mixed 24-hour specimen of urine are treated with 
150 c.c. of 95 per cent, alcohol and the mixture set aside for 48 hours to allow 
the calcium oxalate to precipitate. It is then filtered, each particle of the pre- 
cipitate being transferred to the filter by means of hot water and a rubber- 
tipped glass rod. This precipitate is then washed with hot water and later 
with I per cent, acetic acid. The precipitate is then washed from the fil- 
ter-paper by a stream of dilute hydrochloric acid from a wash-bottle until 
every trace of the precipitate is removed from the filter and dissolved. The 
filter is then washed with hot water until the washings are no longer acid in 
reaction. The hydrochloric acid solution and the washings are evaporated 
to about 20 c.c, a little calcium chlorid solution is added, the solution is neu- 
trahzed with ammonia, is then rendered slightly acid with acetic acid, and 
95 per cent, alcohol added in an amount equal to one-half the volume of the 
liquid. The mixture is then set aside for 48 hours, after which the precipitate 
of calcium oxalate is collected on an ash-free filter, washed with cold water 
and dilute acetic acid until free from chlorids, and the filter with its contents 
is incinerated in a weighed platinum crucible. This latter process is first 
carried out oyer a Bunsen burner and later over a blast-lamp. The crucible 
is then dried in a desiccator and weighed. The difference in weight represents 
the amount of calcium oxid obtained from 500 c.c. of urine. Each gram of 
this oxid represents 1.6 grams of oxalic acid. 

(4) Ferments. 

Several ferments^ have been demonstrated in the normal and pathologic 
urine, but do not seem to have any great clinical importance at the present 
time. 

^ Williams and Williams (Arch. Diagnosis, 1913; VI, 263), who show the relationship of 
obscure pain and hematuria to the excretion of small crystals of calcium oxalate. Fittipaldi, 
Rif. Med., 1915, XXX, 981; Sedgwick, Am. Jour. Dis. Child., 1915, X, 414; Hernando, 
Siglo Med., 1917, LXIV, 50; Fittipaldi, Rif. Med., 1917, XXXII, 1173. . 

2 Jour. Exper. Med., 1903, V, 27. 

^ See Lindemann, Ztschr. f. klin. Med., 191 2, LXXV, 58; Kahn and 'Brim, Am. Jour. 
Obs. and Dis. Women and Child., 1915, LXXI, 39, have studied the urinary catalase but 
find nothing of diagnostic value. 



THE URINE 265 

Pepsin. 

This ferment is present in practically every specimen of urine. It has 
been found by Grober, Gehrig, Griitzner, Mathes, Stadelmann, and others. 
It seems to be absent or very much diminished in cases of typhoid fever, 
gastric carcinoma, and hypoacidity.^ In cases of pneumonia, Lenobel and 
Kun and Lochbihler have observed the presence of a ferment, which seemed 
to be pepsin in increased amounts. Scola reports a diminution of the normal 
pepsin content in severe diseases of the nervous system. 

Diastase. 

Traces of this ferment are found in normal urine. It is increased by a 
carbohydrate-rich diet and diminished by a carbohydrate-poor regime. 
Wohlgemuth^ believes an increase is associated with a disturbance of pan- 
creatic function. It is lessened in nephritis. In diabetes the diastase varies 
with the sugar output. 

Lipase. 

This ferment is present normally only in minute traces. It is found, 
however, in cases of hemorrhagic pancreatitis, jaundice, and in diabetes melli- 
tus. It may be detected by the method of Kastle and Loevenhart which is as 
follows: In each of three flasks are placed 5 c.c. of urine. One of the flasks 
is boiled to destroy the ferment which may be present. To a second flask are 
added a few drops of phenolphthalein solution and the acidity determined 
by titration with tenth-normal sodium hydrate solution. The amount of 
alkali necessary to neutralize the 5 c.c. of urine is then added to each of the 
other flasks. To these flasks are then added 0.25 c.c. of ethyl-butyrate and o.i 
c.c. of toluol, the flasks being then placed in the incubator at 37°C. for 24 
hours. To each of these flasks there is then added J^ c.c. more tenth-normal 
hydro chloric acid than the amount of tenth-normal alkali previously added. 
The mixture is then shaken out with 50 c.c. of ether and 25 c.c. of alcohol 
to remove the butyric acid. This is then titrated with tenth-normal 
sodium hydrate, each c.c. of tenth-normal sodium hydrate representing 
0.0088 gram of butyric acid.^ 

(5) Mucin-like Substances. 
(a) Mucin. 

True mucin is present in traces in practically all urine. It is found both 
as an insoluble portion which forms the nubecula and as a soluble portion 
which is much smaller in amount than the insoluble form, is precipitated by 

1 See von Kozawa, Internat. Beitr. z. Path. u. Ther., d. Ernahrungsstor., 191 2, IV, 44; 
Fuld and Hirayama, Ztschr, f. exp. Path. u. Ther., 191 2, X, 248; also, Tachau, Ztschr. f. 
klin. Med., 1912, LXXVI, 167; Okada, Mitt. a. d. Med. Fakultat, Tokio, 1914, XI, 293; 
Fernandex-Arroyo, Siglo Med., 1916, LXIII, 435. 

2 Biochem. Ztschr., 1909, XXI, 432; Berl. klin. Wchnschr., 1910, XL VII, 92 and 1444. 
See, also, Corbett, Jour. Obst. and Gynec. Brit. Emp., 1913, XXIII, 227; Neumann, 
Deutsch. Arch. f. klin. Med., 1913, CXI, 164; and Geyelin, Arch. Int. Med., 1914, XIII, 96; 
Marino, Semana Med., 1914, XXI, 907; Lister, Brit. Med. Jour., 1914, II, 584; Brown and 
Smith, BuU. Johns Hopkins Hosp., 1914, XXV, 213; King. Am. Jour. Physiol., 1914, XXXV, 
301; Kahn and Brim, Am. Jour. Obs., 1915, LXXI, 39; Rowntree, Marshall and Baetjer, 
Arch. Int. Med., 1915, XV, 543; Stocks, Quart. Jour. Med., 1916, IX, 216; McClure and 
Pratt, Arch. Int Med., 191 7, XIX, 568. 

2 See Quinan, Jour. Med. Research, 1915, XXXII, 45. 



266 DIAGNOSTIC METHODS 

acetic acid, but is easily soluble in a slight excess of the acid. This form of 
protein is derived from the urinary passages and has practically no pathologic 
importance, although it may be much increased in catarrhal conditions of the 
urinary tract in which it may appear as a gelatinous ropy material, or in rare 
cases in the form of casts of the ureter or urethra from i to lo cm. long and 3 
to 4 mm. thick. These cases are rare and have been reported by von Jaksch 
under the name of ''ureteritis membranacea" and by Frank under the 
name of "pyelitis productiva." 

Mucin is precipitated by the ordinary reagents for albumin (to be dis- 
cussed later), but is soluble in an excess of these reagents, so that it need not 
be mistaken for albumin. It is frequently confused with the ''nucleo-albu- 
mins," but may be distinguished, chemically, by the fact that it contains no 
phosphorus and gives on heating with acids a substance which reduces 
copper solutions. 

(b) Nucleo-albumin. 

The large majority of specimens of urine contain a substance (other than 
true mucin) which is precipitated on the addition of cold acetic acid. Dilute 
acetic acid does not dissolve the precipitate, so that true mucin is excluded, 
as the latter is usually dissolved even by dilutions of acetic acid which do not 
precipitate the other bodies. The reaction with acetic acid is somewhat 
intensified if the urine be diluted. 

Practically every normal urine contains traces of such substances, which 
will give a precipitate with acetic acid, especially if the salts be removed by 
dialysis. In all probability this body, found in normal urines, is either euglo- 
bulin or a mixture of this protein with fibrinogen. It has been called '' nucleo- 
albumin," but this is in all probability a misnomer. It is probably true that 
real nucleo-albumin is never a normal constituent of the urine. In this con- 
nection it must be stated that much confusion exists regarding the nature of 
true nucleo-albumin.^ This substance has been considered to be identical 
with nucleo-protein, but with absolutely no basis of chemical facts. Nucleo- 
protein (true nuclein) is a combination of protein with the prosthetic group, 
nucleinic acid, which splits up into phosphoric acid and purin bodies; while 
nucleo-albumin (pseudo-nuclein) is a combination of protein with paranu- 
cleinic acid, which is rich in phosphorus, but does not yield purin bases on 
hydrolysis. 

Morner^ beheves that most of the so-called "nucleo-albumin" is a com- 
pound of true serum-albumin with an albumin-precipitating body formed on 
addition of acetic acid. He showed that there were three such precipitating 
bodies present in the urine; chondroi tin-sulphuric acid^ was practically al- 
ways present, nucleinic acid occasionally present, and tauro-cholic acid, which 
may be normally present in traces, but in certain pathologic conditions is 

^ See Jones, "Nucleic Acids," Longmans Green & Co., New York, 1914. 

2 Skand. Arch. f. Physiol., 1895, VI, 332. 

3 See Levene and La Forge, Jour. Biol. Chem., 1913, XV, 69 and 155; also, PoUitzer, 
Deutsch. Med. Wchnschr., 191 2, XXXVIII, 1538; Med. Klin., 1913, IX, 2101; Levene and 
La Forge, Jour. Biol. Chem., 1914, XVIII, 123; Ibid., 1915, XX, 433; Levene and L6pez- 
Sudrez, Ibid., 191 8, XXXVI, 105. 



THE URINE 267 

much increased. He believes that these precipitating bodies are normally 
present in excess and, therefore, any increase of a precipitate on addition 
of the acetic acid would mean an increased excretion of albumin. The 
more these precipitating bodies predominate the more the precipitate resem- 
bles "nucleo-albumin." 

From the pathologic standpoint what has been called true ''nucleo- 
albumin" appears in conditions destroying the integrity of the epithelium of 
the uriniferous tubules or of the bladder as well as in conditions associated 
with the excretion of pus in the urine. Thus we would expect to find such a 
body in acute nephritis, whether the result of bacterial or of exogenous toxins, 
in the acute febrile diseases, in renal hyperemia, in leukemia, in acute yellow 
atrophy, and in obstructive jaundice, in which case this body is derived 
doubtless from the bile. In cases of nephritis this body, precipitable by acetic 
acid in dilute solution, may precede and follow the true albuminuria In 
amyloid kidney this body seems to be the chief type of protein present. In 
orthostatic albuminuria this substance may be the only protein present and 
may persist after the others have cleared up. In cases in which the urine 
contains a large number of epithelial cells, casts, and pus-cells, Matsumoto finds 
a substance precipitable by acetic acid, but only in very small amounts. This 
finding would seem to indicate that nucleo-albumin, if such ever occurs, is at least 
not an indication of cellular origin from increased epithelial desquamation. 
This body may be found in general catarrhal conditions of the urinary tract, as 
in cystitis or pyelitis, but in such cases we are more apt to obtain true mucin. 

In the above discussion the writer has not attempted to differentiate these 
bodies, as the reports in the literature have little importance beyond the fact 
that a body precipitable by acetic acid was obtained. Each worker has named 
this body as he understood it and in many cases has had no definite basis for such 
conclusion. To remove it from the urine, add a solution of lead acetate 
and filter; the precipitated phosphates and chlorids carry down this protein. 

(6) Pigments and Chromogens. 
{a) Urochrome. 

This pigment is the chief coloring matter of normal urine, imparting a 
yellow, orange, or a brownish color to the urine, depending upon its concen- 
tration. It is closely related to urobilin, especially the so-caUed normal 
urobilin of MacMunn, as this latter body may be readily converted into uro- 
chrome by evaporation of its aqueous ethereal solution. It is, in aU prob- 
ability, aj mixture of one or more pigments, contains about 4 per cent, of 
nitrogen, and is free from iron. It is readily soluble in water and alcohol; 
sparingly soluble in acetic ether, amyl alcohol, and acetone; insoluble 
in ether, chloroform, and benzol. 

An increase of urochromxOgen, the precursor of this pigment, seems to be of 
some clinical importance. Weisz^ has introduced the following test for its 

1 Med. Klin., 1910, VI, 1661; Biochem. Ztschr., 191 1, XXX, 333; Wien. klin. Wchnschr., 
1912, XXV, 1183 See, also, Heflebower, Am. Jour. Med. Sc, 1912, CXLIII, 221; Paranhos 
and Giolito, Brazil-med., 1913, XXVII, loi; Vitry, Rev. internat. de la tuberc, 1913, 
XXIII, 325; Skorczewski, Ztschr. f. exper. Path. u. Therap., 1913, XIV, 113; Salustri, 
Policlinico, 1913, XX, 1729; Nicola, Gazz. d. osp., 1913, XXXIV, 1465; Keim and Vigot, 



2 68 DIAGNOSTIC METHODS 

detection. Place the suspected urine in a test-tube and dilute with three 
times its volume of water. Mix well and add 3 drops of a i to 1000 aqueous 
solution of potassium permanganate. Shake well. A yellow tint or an 
increase in the yellow tone of the mixture indicates a positive reaction. This 
reaction is an indication of a toxic metabolic disturbance and is given in cases 
with high fever, especially in pulmonary tuberculosis and typhoid fever. It 
is never seen normally and, rarely, in carcinoma. A positive reaction is a 
contra-indication to the use of tuberculin in tubercular cases. 
(b) Uroerythrin. 

This pigment is a constituent of a large majority of normal urines, al- 
though it is probable that it indicates a slight deviation from the normal. It 
has been called rosacic acid by Prout and purpurin by Golding-Bird. To 
this pigment is due the salmon or brick-red color which the urinary sediments 
take in highly concentrated febrile urines. Normally, it may not be present 
in sufficient amount to color the urine, but under pathologic conditions it may 
impart a deep orange tint to this fluid. It is soluble in amyl alcohol, slightly 
soluble in acetic ether and absolute alcohol and very difficultly soluble in water. 

This pigment appears to be increased on a meat diet, after severe exercise, 
profuse perspiration, or by irregular digestion. Pathologically, it is observed 
especially in cases of hepatic insufficiency, in chronic cardiac and pulmonary 
disease, in acute articular rheumatism, in malarial fever, and in general acute 
febrile diseases. In typhoid fever one does not find this pigment as fre- 
quently as in most other acute febrile conditions. 
(c) Urobilin. 

This substance appears in the urine not as a free pigment, but in the form 
of the chromogen urobilinogen, which is decomposed into urobilin through the 
influence of the light. It is claimed that various types of urobilin are found, 
as for instance the normal urobilin of MacMunn and the pathologic urobilin 
of Jaffe. Whether these are really different bodies is at present an un- 
settled question. Urobilin appears to be identical with the stercobilin of 
the feces and is not the same as the normal fecal hydrobilirubin. Much 
discussion has centered around the origin of this pigment. Passing through 
the stages of the hepatogenous, hematogenous, nephrogenous and histogene- 
tic urobilinuria, the general concensus of opinion seems at present to be that 
most of the urinary urobilin is of enterogenous origin.^ According to this 
theory, the pathogenesis of urobilinuria may be presented as follows: 

Presse med., 1914, XXII, 153; Martelli and Pizzetti, Policlinico, 1914, XXI, 182; Gullbring, 
Hygiea, 1914, LXXVI, 599; Metzger and Watson, Jour. Am. Med. Assn., 1914, LXII, 1886; 
Schaffle, t d., 1914, LXIII, 1294; Pignacca, Gazz. d. osp., 1914, XXXV, 353; Tuliato, 
Ibid., 665; Bal jzzi and Ballero, Ibid., 2053; Rhein, Miinch. med. Wchnschr., 1914, LXI, 
2355; Bruni, Gazz. d. osp., 1915, XXXVI, 401; Ferrannini, Riforma Med., 1915, XXXI, 
477; Halbey, Med. Klin., 1915, XI, 833; Miihlens, Miinch. med. Wchnschr., 1915, LXII, 
1067; Pulay, Ibid., 1009; Genoese, Policlinico, 1915, XXII, 558; Biesenthal, 111. Med. 
Jour., 1915, XXVIII, 344; Pulay, Miinch. Med Wchnschr., 1915, LXII, 1009; Svestka, 
Wien. klin. Wchnschr., 1915, XXVIII, 1054; Burgess, Jour. A. M. A., 1916, LXVI, 82; 
Cowen, Ibid., 791; Belgrano, Policlinico, 1916, XXIII, 276; Schwensen, Ugeskrif t f . Laeger, 
191 7, LXXIX, 231; Lollini, Policlinico, 191 7, XXIV, 308. 

^ See Wilbur and Addis, Jour. Am. Med. Assn., 1912, LIX, 929; also, Fromholdt and 
Nersessofif, Ztschr. f. exper. Path. u. Therap., 191 2, XI, 400; Wilbur and Addis, Arch. Int. 
Med., 1914, XIII, 235; Feldner, Centralbl. f. d. Grenzgeb. d. Med. u. Chir., 1915, XIX, 163. 



THE URINE 269 

"The liver-cell, both in normal and abnormal conditions, forms only 
bilirubin from the blood pigment. Providing there is no marked obstruction 
to the passage of bile into the intestine, the bilirubin is acted upon by bacteria 
whicli reduce it so completely to urobilin that only traces of bilirubin appear 
in the feces. A part of the urobilin is absorbed and is excreted in the urine, 
while traces appear in the bile and in pathologic transudates. When bacterial 
action is excluded, as in the new-born, no urobilin is found in the urine. 
Further, when bile is not present in the intestine, as in cases of absolute oc- 
clusion of the ductus choledochus, urobilinuria does not occur. It is sparingly 
excreted when the production of biliary pigment is diminished, as in hunger, 
while the amount is small or at most normal in cases of incomplete exclusion 
of bile from the intestine. On the other hand, the amount excreted may 
reach abnormal limits if a preceding obstruction, accompanied by stasis, has 
been overcome, and bile flows freely into the intestine. Likewise the quantity 
may be abnormally large if the production of biliary pigment from the red 
blood-corpuscles increases as a result of infection and intoxication, or of he- 
patic lesions, such as cirrhosis and cyanotic induration. In these cases the 
bile is tenacious and the condition may give rise to jaundice although it is sel- 
dom that the stasis is so great that the bile is completely shut off from the in- 
testine. Indeed, in most cases, owing to the excretion of excessive pigments in 
the bile (pleiochromia), a more than normal amount of pigment passes into the 
intestine, and as a result of this there arises a marked urobilinuria with a mild 
degree of biliary stasis. In many cases the stagnation of bile is great enough 
to cause a passage of biliary pigment from the blood into the urine, leading to 
a marked urobilinuria, a mild degree of bilirubinuria, and a yellow- tin ting of 
the tissues. In other cases the absorption of bile is so slight that only" yellowing 
of the tissues results, yet the concentration of pigment in the blood does not 
suffice to permit of its excretion by the kidneys, so that marked urobilinuria 
and yellowing of the tissues without bilirubinuria ensues" (Weintraud). 

Urobilin is found in febrile conditions, chronic passive congestion, lead- 
poisoning, cases in which extravasation of blood into the tissues occurs, in 
any condition associated with marked hemolysis, hepatic cirrhosis, and in 
the cases of jaundice outlined above by Weintraud. It has been noticed in 
increased amounts in Addison's disease, extrauterine pregnancy, hemophilia, 
and in secondary syphilis.^ 

The presence of an increased amount of urobilin usually causes a dark- 
yellow color of the urine, the foam in such cases being colored, more perhaps 
due to the presence of other pigments than to urobilin itself. 

Urobilin is soluble in ethyl alcohol, amyl alcohol, and chloroform, and 
slightly soluble in ether, acetic ether, and in water. If an acid solution of 
urobilin be examined with a spectroscope, it shows a broad absorption band 

^ See Simon, Med. Klin., 1913, IX, 1164; Hildebrandt, Arch. f. Verdauungskr., 1913 
XIX, 442; Kamsarakau, Med. Obozr., 1913, LXXIX 559; Molnar, Virchow's Arch. f. path 
Anat., 1913, CCXIII, 433; Hausmann, Ztschr. f. exper. Path. u. Therap., 1913, XIII, 373 
Litzenberg, Am. Jour. Obs. and Dis. Women and Child., 1916, LXXIII, 228; Kirch 
Wien. klin. Wchnschr., 1916, XXIX, 1238; Barnard^ Jour.-Lancet, 1917, XXXVII, 80: 
Boyd, Jour. Lab. and Clin. Med., 1919, IV, 495; Hausmann and Howard, Jour. A. M. A., 
1919, LXXIII, 1262. 



270 ' DIAGNOSTIC METHODS 

to the right of E, the left-border of which reaches nearly to h, while the right 
border encloses F. If the solution be alkaline, the spectrum shows a l^s 
broad absorption band between E and F enclosing h. This solution is 
best made as follows: Ten to 20 ex. of the urine are acidulated with a 
few drops of hydrochloric acid and shaken out with from 6 to 10 c.c. of 
amyl alcohol, which then shows the characteristic spectrum of acid urobilin. 
If to a small portion of this amyl alcohol solution be added a few drops of i per 
cent, solution of zinc chlorid, which has been strongly alkalinized with am- 
monia, a beautiful green fluorescence appears.^ With Ehrlich's dimethyl- 
amido-benzaldehyde reaction (p. 354) this pigment gives a distinct red 
color. 

{d) Indican.2 

In the decomposition of protein occurring in the intestinal canal indol 
(CsHyN) and skatol (C9H9N) are found among the products of this bacterial 
cleavage. These substances are absorbed and oxidized in the blood to indoxyl 
(CsHtNO) and skatoxyl (C9H9NO). These bodies are then conjugated with 
sulphuric acid forming indoxyl and skatoxyl sulphuric acids, after which 
they are excreted in the form of the potassium salt, indoxyl potassium sul- 
phate (CgHeNO — SO2 — OK) and skatoxyl potassium sulphate (C9H8NO — 
SO2 — OK) . To the former of these is given the name indican. This chromo- 
gen is present in much larger amounts than is the skatoxyl compound so that 
our discussion will include principally the former. 

The absolute amount of indican occurring in the urine depends upon the 
amount of decomposition occurring in the intestine. The largest amounts are 
naturally observed in health following a meat diet, while the urine may be 
practically free from indican on a vegetable or a milk diet. The pathologic 
variations may be summed up as follows. An increased elimination of indican 
is observed in all diseases which are associated with an increased intestinal 
decomposition.^ This decomposition usually occurs in the large intestine, but 
may at times take place in the small bowel, in which cases the degree of in- 
dicanuria may be greater owing to the increased absorptive power of the 
small bowel. Although many writers state that indicanuria is not seen in 
cases of simple constipation, the writer must take exceptions to these state- 
ments as some of his most intense and persistent cases of indicanuria have 
been found in uncomplicated constipation. Secondly, an increased excretion 
of indican is observed in cases showing diminished peristalsis, as, for instance, 
in ileus and peritonitis. These cases frequently show an intense reaction.'* 

^ See Edelmann, Wien. klin. Wchnschr., 191 5, XXVIII, 978; Marcussen and Hansen, 
Jour. Biol. Chem., 1918, XXXVI, 381. 

2 See Baar, Die Indicanurie, Wien, 191 2; New York Med. Jour., 1914, XCIX, 669. 

^ See Morgan, Am. Jour. Med. Sc, 1912, CXLIV, 827; also, Benedict, Arch. Diagnosis, 
1912, V, 159; Sherwin and Hawk, Biochem. Bull, 1914, III, 416; Jour. Am. Chem. Soc, 
1914, XXXVI, 1779; Hoppe-Seyler, Deutsche Med. Wchnschr., 1916, XLII, 1213.^ 

^ '' Although there appears to be no definite relation of indicanuria and albuminuria, the 
writer has rarely failed to observe large numbers of casts in urines showing marked indican 
reactions. Whipple, Rodenbaugh and Kilgore (Jour. Exper. Med., 1916, XXIII, 123), 
Whipple, (Jour. A. M. A., 1916, LXVII, 15) and Whipple and Cooke (Jour. Exper. Med., 
191 7, XXV, 461 and 479) have shown that the intoxication in intestinal obstruction is due 
to a primary proteose. See, however, Dragstedt, Moorhead, and Bursky, Proc. Soc. 
Exper. Biol, and Med., 1916, XIV, 17. 



THE URINE - 271 

If the obstruction leading to ileus be in the large bowel, indican is either absent 
or appears much later than is the case if the small bowel be involved. Lastly, 
in any condition associated with protein decomposition anywhere in the 
system, as, for instance, in empyema, putrid bronchitis, abscess formation, 
etc., indican may be much increased. 

The color of the urine is usually normal when voided, although a large 
amount of indican may be present. In some cases oxidation of this chro- 
mogen has occurred within the system and the urine appears greenish or 
even blue when voided. If the urine be allowed to stand until decomposition 
occurs, a reddish or bluish metallic-like scum may be observed, due to the 
conversion of the indican into indigo-blue. Most of the tests for the presence 
of indican in the urine depend upon the oxidation of the indican, according 
to the following equation (this body having been previously decomposed by 
concentrated HCl into indoxyl and sulphuric acid). 

2C8H7NO + 2.0 = C16H10N2O2 + 2H2O. 

Tests for Indican. 
Jaffe^s Test. 

A few c.c. of urine are treated with an equal volume of concentrated 
hydrochloric acid, two or three drops of a strong solution of calcium hypo- 
chlorite (bleaching powder or ''chlorid of lime") are added an,d the contents 
mixed. Two c.c. of chloroform are then added and the tube inverted several 
times. In this process the indican is oxidized to indigo-blue which is taken 
up by the chloroform. The depth of the blue coloration of the chloroform 
will serve as an approximate estimate of the amount of indican present. In 
absolutely normal urine no blue coloration or, at most, a faint bluish tinge is 
observed. Care must be taken in this test to avoid an excess of the hypo- 
chlorite, as this will convert the indigo-blue into isatin which is distinctly 
yellow, according to the following equation: 

C16H10N2O2 + 02= 2C8H5NO2. 

Obermayer's Test. 

A few c.c. of urine^ are mixed with an equal volume of Obermayer's reagent 
(a 0.2 per cent, solution of ferric chlorid in concentrated hydrochloric acid) 
and the solutions mixed by repeatedly inverting the tube. A few c.c. of 
chloroform are then added and the tube inverted as before. The presence 
of any appreciable amount of indican is indicated by a dark brown to black 
coloration of the urine and the later absorption of this color by the chloro- 
form, which becomes a more or less deep shade of blue. If the urine be very 
dark in color or if bile pigment be present, the pigments may be removed by 
adding a solution of lead subacetate and filtering. If potassium iodid be 
present this test yields a red color wh'ch will disappear on addition of sodium 
thiosulphate solution. Salicyluric acid gives a violet coloration. If formalin 
be used as a preservative, indican is incapable of detection by any of the tests 
given. Thymol-containing urines give a violet coloration to the chloroform 

^Askenstedt, Jour. Lab. and Clin. Med., 1917, II, 578, advises warming the urine 
before addition of the reagent. 



2 72 DIAGNOSTIC METHODS 

extract. This color is destroyed by sodium hydroxide or thiosulphate.^ 
Jolles^ has made this fact the basis of a new test for indican in the urine. 

Instead of using the Obermayer reagent as such, the urine may be mixed 
with the hydrochloric acid and one or two drops of a lo per cent, solution of 
ferric chlorid may then be added to the mixture. This test is a much more 
reliable one than is the Jaffe test, as the latter is very prone to carry the 
oxidation to the isatin stage rather than to the indigo-blue phase. ^ 

Skatoxyl-potassium Sulphate. 

A certain amount of skatoxyl-potassium sulphate is formed along with 
the indican. In some cases for reasons not well understood, this pigment or 
one closely allied to it appears in the urine in excess of the indican, so that 
the above test may give distinct red colorations of the chloroform instead 
of the usual blue.^ Just exactly what pigment causes these red variations is 
doubtful. Some of this pigment is always present along with the indican 
and may be extracted either with hot water or mth a mixture of alcohol, 
ether and water. To this red pigment has been given the name indigo-red, 
skatoxyl-red, urorubin, urorhodin, and several others. 

If the urine be heated on applying Jaffe's test, a dark red coloration is 
observed, while if Obermayer's test be' used the coloration will be of a 
reddish-violet. 

Rosenbach's Test. 

A few c.c. of urine are boiled and concentrated nitric acid added drop by 
drop during the boiling. The urine takes a deep red color and the foam ap- 
pears bluish-red. If the nitric acid be much in excess the urine will assume a 
yellowish-red color and the foam a distinctly yellow tint. If sodium hydrate 
or ammonia be now added drop by drop, a bluish-red precipitate is observed 
which is soluble in an excess of the alkali with a brownish-red coloration. 
This test is due to the presence of indigo-red. Its clinical significance is the 
same as that of indican. This pigment may be obtained directly from the 
urine by neutralizing it with sodium hydrate and shaking out with ether, 
when the ether will take a distinct red color. 

Quantitative Determination. 
Wang^s Method. 

The principle of this method is the decompostion of indican and its 
oxidation to indigo-blue. This compound is then transformed into indigo- 
sulphuric acid, which is directly determined by titration with potassium per- 
manganate solution. 
Technic. 

A preUminary determination of the relative amount of indican is made 
with Obermayer 's reagent. If a strong reaction is obtained, from 25 to loo c.c. 

1 Rosenbloom, New York Med. Jour., 1913, XCVIII, 814. 

2 Ztschr. f. physiol. Cham., 1913, LXXXVII, 310. See, also, Stanford, Ibid., 188 and 
Ibid., 1913, LXXXVIII, 47; also, Edes, Arch. Diagnosis, 1913, VI, 152; Rossi, Policlinico, 

1914, XXI, 297;Cantelli, Gazz. d. osp., 1915, XXXVI, 833; Jolles, Ztschr. f. physiol. Chem., 

1915, XCIV, 79; Ibid., 1915, XCV, 29. 

3 See Natonek, Zentralbl. f. inn. Med., 1913, XXXIV, 1124. 
'* See de Jager, Nederl. Tijdschr. v. Geneesk., 1916, II, 1873. 



THE URINE 273 

of urine are used, while if the reaction be sHght 200 to 500 c.c. are necessary. 
The urine should be acidified with acetic acid, unless its reaction be already acid. 

Fifty c.c. of urine, or a larger amount if the conditions mentioned 
above obtain, are treated with 5 c.c. of a 20 per cent, solution of lead 
acetate, or one-tenth the volume of urine in case a larger amount of urine 
be taken. The urine is then filtered and a large and accurately measured 
portion of the filtrate is treated, in a separatory funnel, with an equal 
volume of Obermayer's reagent. This mixture is then shaken out with 
chloroform, using 30 c.c. of this menstruum and shaking for one minute. 
At least four such chloroform extractions should be made, more if the chloro- 
form still extracts indigo-blue from the mixture. The chloroform extract 
is placed in a small flask and the chloroform distilled. The residue in the 
flask is dried for a few minutes on the water-bath to remove the last traces 
of the chloroform, and is washed either with hot water or with a mixture 
of equal parts of alcohol, ether, and water. These solvents remove the red 
coloring matter, leaving the indigo-blue undissolved. The extract is filtered 
through a small filter and the indigo-blue completely transferred to the 
filter, after which it is thoroughly washed with hot water. This indigo- 
blue is dissolved on the filter with boiling chloroform, the filtrate being 
allowed to run into the original flask. The chloroform is again distilled 
and the residue dried on the water-bath. This purified indigo-blue is dis- 
solved in 10 c.c. of concentrated sulphuric acid and the solution diluted to 
100 c.c. with water. 

This solution of indigo-sulphuric acid is then titrated with standard potas- 
sium permanganate solution of such a strength that i c.c. will represent 
approximately 0.0062 gram of indigo-blue. This solution of potassium per- 
manganate contains about 3 grams of potassium permanganate to the liter. 
Its titer is determined before use by titrating against a solution of pure 
indigo-blue in sulphuric acid. In making this titration the concentrated solu- 
tion is not used, but a dilute one made by diluting 5 c.c. of the stronger 
permanganate solution to 200 c.c. (each c.c. of which will represent 0.000155 
gram of indigo-blue). In this titration the bliie color of the indigo-sul- 
phuric acid does not change to any extent on the addition of the first drops 
of the permanganate solution, but gradually turns greenish and then becomes 
yellowish or entirely colorless. The amount of indigo-blue in the urine used 
is readily ascertained by multiplying the number of c.c. of permanganate 
solution used by the amount of indigo-blue represented by the titer of the 
solution (in the writer's laboratory i c.c. of the diluted solution equals 
0.000155 gram of indigo-blue). 

It has been found by Ellinger that only about 87 per cent, of the theoret- 
ical yield is obtained by this method, owing, probably, to the simultaneous for- 
mation of isatin from the indigo-blue. The average daily excretion of indi- 
can, as revealed by this test, ranges between 5 and 20 mg. 
Folin's Method. 

This method has not, as yet, been extended so as to give absolutely quan- 
titative results. Its principle is the comparison of the color given when urine 
18 



274 DIAGNOSTIC METHODS 

is treated with Obermayer's reagent with that of FehHng's solution as a stand- 
ard. To this standard has been given the arbitrary value of loo. 

Exactly one one-hundredth of the 24-hour specimen of urine is taken for 
each determination and treated with an equal volume of Obermayer's re- 
agent. The indigo-blue is then extracted with 5 c.c. of chloroform until all 
of the pigment has been dissolved. With the chloroform solutions are then 
made colorimetric comparisons with FehUng's solution, using either the Du- 
boscq or Sargent instrument. Folin finds the average indican excretion, on 
this basis, from a diet of 119 grams of protein to be 77, while on a nitrogen- 
free diet no indican is found. 

(e) Uroroseinogen. 

This chromogen, shown by Herter^ to be indol-acetic acid, is converted by 
oxidation into the pigment urorosein, which is soluble in ethyl and amyl alco- 
hol but insoluble in chloroform, ether and benzol. If the urine stands for 
some time bacterial action produces this pigment, although an alkaline reac- 
tion may destroy the coloration. Its amyl-alcoholic solution shows a sharp 
narrow absorption band between D and E.^ Although this pigment appears 
in normal urine only in traces, it may be markedly increased by a strict vege- 
table diet. Pathologically, it appears in cases of severe gastric and intestinal 
disturbances, tuberculosis, pernicious anemia, nephritis, severe chlorosis, 
diabetes mellitus, carcinoma, osteomalacia, typhoid fever and dementia 
praecox.2 It has, therefore, little differential value although it is, often, as 
significant as indican of excessive protein decomposition in the bowel. 

Test for Urorosein (indol-acetic acid) : If 5 c.c. of urine be mixed with 
5 c.c. of chemically pure HCl and a few drops of a i per cent, solution of 
sodium nitrite a characteristic rose-red coloration develops, which is due to 
oxidizing rather than nitrifying action. This pigment also gives a red color- 
ation with Ehrlich's dime thy 1-amidobenzaldehyd reaction. 

(B) Abnormal Composition. 
(i) Proteins. 

There has been much discussion as to whether a true physiologic pro- 
teinuria occurs.^ Without going into a discussion of the subject the writer 
wishes to state his behef that a true physiologic proteinuria occurs, but that 
such may never be detected by the usual clinical methods of examination. 
For our purposes, therefore, the presence of protein of one type or another in 
amounts amenable to detection by clinical methods must be considered 
pathological. 

(a) Senmi Albumin. 

From the pathologic standpoint, serum albumin is the most important 
protein found in the urine. The amount excreted in 24 hours is variable and 
does not necessarily have any relation to the severity of the kidney lesion 
should one reaUy exist. From 5 to 10 grams of this protein per diem may be 

^ Jour. Biol. Chem., 1908, IV, 107, 239 and 253. 

2 See Riesser, Inaug. Dissert., Konigsberg, 191 1. 

' See Ross, Arch. Int. Med., 1913, XII, 112 and 231. 

^ See Backman, C. R., soc, biol., Paris, 1916, LXXIX, 339. 



THE URINE 275 

regarded as a moderate pathologic excretion , while a lesser would be of little 
significance and a greater would be regarded as excessive. As large amounts 
as 40 grams have been observed, but such findings are exceptional. Morner 
considers an excretion of albumin varying from 2 5 to 75 mg. per liter as a nor- 
mal output, which amount would, however, escape the usual clinical tests. 

As some of the more delicate tests for albumin, as for instance Spiegler's 
reagent, will show traces of albumin in practically every specimen of urine 
examined, we should Hmit our conception of albuminuria to those cases which 
react with the ordinary tests rather than with the most delicate. According 
to Hofmeister, the standard upon which one bases a judgment as to the pres- 
ence of a pathologic albuminuria is the formation of a distinct albumin ring 
within three minutes after the urine and nitric acid are in contact in Heller's 
test (see below) . Moreover, the term albuminuria should be limited to those 
cases in which there is some disturbance of the renal epitheUum, especially of 
the glomeruH. This does not exclude those cases of purely functional albu- 
minuria in which no distinct lesion of the kidney exists, as these cases are 
all associated with some abnormahty of the excreting organ. 

It not infrequently happens that albumin, as found in the urine, is derived 
from some portion of the urinary tract below the kidney, as when inflamma- 
tory exudates, blood, lymph, spermatic or prostatic fluids, pus and other ex- 
traneous material are mixed with the urine after it is excreted by the kidney.^ 
To such cases is given the nsime false or accidental albuminuria, in contra-dis- 
tinction to the true albuminuria in which the albumin is present when ex- 
creted by this organ. Before reporting a finding as one of true albuminuria, 
it is absolutely imperative that extraneous albumin be excluded. 
Functional Albuminuria. 

Not infrequently do we find in perfectly healthy individuals a urine which 
is normal in every way with the exception of the presence of albumin, which 
is easily detected by our ordinary methods. This type of albuminuria is 
observed following severe muscular exercise beyond the point to which the 
subject is accustomed. Thus, in raw recruits under the severe forced marches 
during the early days of their service we find this type very prevalent. Leube 
states that 59 per cent, of such soldiers show a temporary albuminuria, which 
disappears after the subject becomes accustomed to the increased exercise. ^ 
It is a common occurrence to find such a functional albuminuria in football 
players, bicycle riders, crew men, and general athletes after every period of 
increased exertion. This question may be summed up by the statement that 
the excretion of albumin is simply dependent, under these conditions, upon 
the limit of endurance of the subject, practically everyone being able to pro- 
duce an albuminuria if he overdoes in any way. Moreover, cold baths, excess- 
ive mental labor, and severe emotion may lead to quite an extensive albumin- 

^ See Belfield (Jour. Am. Med. Assn., 1915, LXIV, 2040) for a discussion of vesicular 
albuminuria due to presence of both globulin and albumin derived from the seminal vesicles. 

2 See von Hecker, Berl. klin. Wchnschr., 1913, L, 1848; Bugge, Norsk Mag. f. Laegevi- 
densk., 1913, LXXIV, 1601; Reber and Lauener, Corr.-Bl. f. Schweiz. Aerzte, 1915, XLV, 
949; Mossy and Richet, Paris Medical, 191 7, VII, 47; Bornstein and Lippmann, Ztschr. f. 
klin. Med., 1918, LXXVI, 344; MacLean. Jour. Royal Army Med. Corps, 1918, XXXI, 
245 and 319. 



276 DIAGNOSTIC METHODS 

uria, especially in an individual with somewhat lessened resistance. Ac- 
cording to Rem-Picci albuminuria is a constant finding after cold baths, diff- 
erent subjects reacting differently to the same stimuli. This albuminuria 
never lasts over 24 hours and may be associated with the appearance of casts 
and blood, as may also the type following increased exercise. The colder the 
bath and the longer the immersion, the more rapid the appearance of albumin. 

Another type of functional albuminuria is that following the intake of a 
heavy protein meal. This is known as ^^ alimentary albuminuria.^' It has 
been supposed to be due to filtration from the blood, the foreign protein being 
absorbed and excreted without interv^ening hydrolytic cleavage. Uhlen- 
huth,^ Inouye,^ and Croftan,^ working with the precipitin test, have found 
egg albumin in the blood and urine in these cases. Ascoli^ later demon- 
strated both serum and egg albumin by this test. Wilson^ using the fixation 
of complement test, found the main element to be of human origin. Recently 
Wells, ^ applying the anaphylaxis reaction,^ has shown that sensitized animals 
do not react to egg albumin but show marked reactions to human protein. 
Owing to this disagreement of the anaphylaxis and precipitin tests, it would 
seem probable that the excreted albumin is derived from the blood rather than 
directly from the food, little if any unaltered food protein ever reaching the 
urine. ^ 

The albuminuria observed in the new-born for the first few days of life, 
aside from the influence of feeding upon foreign proteins, is probably a further 
example of a true functional albuminuria. Likewise, the albuminuria ob- 
served in pregnancy is usually distinctly functional. About 50 per cent, of 
pregnant women show this albuminuria, little difference being observed 
between primiparae and multiparse. The kidney undoubtedly shows some 
functional disturbance in its attempt to eliminate the toxic products 
absorbed from the fetus. ^ 

According to Senator^ ° such albuminurias as outlined above should be con- 
sidered functional when slight in degree, transitory in character, occurring 
after unusual strain, either physical or mental, the subjects showing a further 
negative history after the removal of the direct stimulus. Whether this type 
is to be considered physiologic rests entirely upon the conception of the nor- 
mal or abnormal nature of the stimuli leading to the excretion of albumin. It 

^ Deutsch. med. Wchnschr., 1900, XXVI, 734. 

2 Deutsch. Arch. f. klin. Med., 1903, LXXV, 378. 

3 New York Med. Jour., 1909, LXXXIX, 474. 

^ Miinch. med. Wchnschr., 1903, L, 201 and 1761; Ztschr. f. phvsiol. Chem., 1903, 
XXIX, 283. 

^ Jour. Path, and Bacteriol., 1909, XIII, 484. 

^ Jour. Am. Med. Assn., 1909, LIII, 863. 

^ See Schittenhelm and Weichardt, Ztschr. f. exper. Path. u. Therap., 191 2, XI, 69; 
Pearce, Jour. Exper. Med., 1912, XVI, 349; also, Schloss, Am. Jour. Dis. Child., 1912, III, 
341- 

^ See Van Alstyne and Grant, Jour. Med. Research, 191 1, XXV, 399; also, Van Alstyne, 
Arch. Int. Med., 1913, XII, 372; Salus, Biochem. Ztschr., 1914, LX, i; Cameron and Wells, 
Arch. Int. Med., 1915, XV, 746; Bronfenbrenner, Andrews and Scott, Jour. Am. Med. 
1915, LXIV, 1306; Longcope, Am. Jour. Med. Sc, 1916, CLII, 625; Hoobler. Am. Jour. 
Dis. Child., 1916, XII. 129. 

^ See Bondi, J. and S., Arch. f. Gynak., 1914, CII, 89; Macleod, Jour. Lab. and Clin. 
Med., 1917, 11; 520. 

^° Erkrankungen der Nieren, Wien, 1902. 



THE URINE 277 

would seem to the writer that the term "physiologic albuminuria" would 
better be given up or at least used with great caution, as it is difficult to 
believe that the severe strain put upon the system and kidneys, in particular, 
can be distinctly, physiologic.^ He is inclined to reserve this term for the 
excretion of the minimal amounts of albumin, which cannot be detected by 
the simpler clinical tests, and to regard the types of albuminuria, as discussed 
above, as ^^ functionary (Pavy), or ^^ constitutional (Martius). 

A second group of cases showing albuminuria is observed in which the 
excretion of albumin may persist for a period varying from days to months, 
may disappear for a variable space and then return for another indefinite 
length of time. To this type is given the name ^^intermittent albuminuria.^^ 
This is purely functional, no lesion of the kidney being manifest. The cases 
usually show a history of an acute infection or of an antecedent nephritis as 
the result of such infection. The most frequent cause, however, is an uncom- 
pensated heart lesion which may or may not be associated with a direct renal 
lesion. Not infrequently we find an hereditary intermittent albuminuria in 
those with a distinct neurotic family history. 

The functional albuminuria may, at times, follow a definite course, disap- 
pearing and reappearing with such regularity that it has been styled ^^ cyclic 
albuminuria.''^ In this form the albumin usually disappears from the urine at 
night and when the patient is flat on his back, but reappears during the day or 
when the subject is erect. The terms " orthostatic, orthotic, or postural albumin- 
uria^^ would, therefore, seem to be more appropriate than the former appella- 
tion. This type is dependent, according to Erlanger and Hooker, upon a low- 
ering of the pulse pressure which constantly occurs when the individual 
changes from the recumbent to the erect position. Jehle and, more recently, 
Nothmann believe that this type is due to a lordosis, and style it, therefore, 
lordotic albuminuria.^ Certain pathologic cases, as beginning nephritis, may 
show a cyclic albuminuria which may extend well into the period of recovery. 
In true cyclic cases the negative physical findings would lead us to class the 
condition among the functional albuminurias. If a case of this type becomes 
persistent and casts are frequently found, subsequent cardiovascular changes 
will usually appear, changing the case into one of true nephritis. The albu- 
min in such cases usually appears in the urine after rising and reaches a maxi- 
mum from noon to 3 or 4 o'clock, then gradually declines, disappearing from 
8 to 10 p. M. If the subject changes his habits of life, the cycle of albuminuria 
will also change. Along with the variations in the excretion of albumin, the 

^ See Wolf, Arch. Diagnosis, 191 2, V, 260. 

2 See Hamburger, Wien. klin. Wchnschr., 191 2, XXV, 262; also Frankel (Deutsch. med. 
Wchnschr., 1912, XXXVIII, 1985), who finds the urinary acidity increased in these cases 
and states that, possibly, the albuminuria is due to abnormal production of acid as Fischer's 
theory assumes. See, also, Gomolitsky, Ztschr. f. klin. Med., 1913, LXXVII, 96; Reyher 
(Monatsschr. f. Kinderhkde., 1913, XII, 82), Died (Wien. klin. Wchnschr., 1913, XXVI, 
258) and von Jagic (Ibid., 1556) believe that tuberculosis is the important factor in this 
condition. See, also, Wendenburg, Arch. f. Kinderhkde., 1913, LXII, 34; Jehle, Die 
Albuminurie, Berlin, 1914; Reyher, Pediatria, 1914, VI, i; Steensma, Nederl. Tijdschr. v. 
Geneesk., 1914, LVIII, 248; Groenendijk, Ibid., 885; Barlocco, Gazz. d. osp., 1914, XXXV, 
2021; Fischl and Popper, Jahrb. f. Kinderhkde., 1915, LXXXI; Zondek, Ztschr. f. klin. 
Med., 1915, LXXXII, 78; Jeanneret, Arch, de Med. des. Enf., 1915, XVIII, 461; Hoist, 
Norsk. Mag. f. Laegevid., 191 5, LXXVI, 1377; Mason and Erickson, Am. Jour. Med. 
Sc, 1918, CLVI, 643; MacKay, Can. Med. Assoc. Jour., 1919, IX, 975. 



278 DIAGNOSTIC METHODS 

other urinary constituents fluctuate in the same manner, the sequence being, 
according to Teissier, increase in pigments, albumin, uric acid, and urea. A 
peculiarity of this type of albuminuria is that it may be even diminished by 
exercise and is less, therefore, after a hard day's work, differing from the 
albuminuria in nephritis or in the cardiac types. 

While there has been much discussion as to whether such cases are not 
truly pathological, most of them at any rate must have a certain insufficiency 
of the renal epithelium. This may be due to changes in the circulation f ollow- 
ing^change in posture, but it is rather surprising that increased exercise would 
not work more strongly in this way. Krehl regards these conditions as rela- 
tively harmless as they do not usually show any subsequent history of neph- 
ritis; Broadbent does not believe such cases ever develop actual renal disease; 
while Senator insists that most of them are cases of nephritis. The patients 
showing this type of albuminuria are weak anemic individuals about the age 
of puberty, subject to fainting spells, with a heart showing intermittent at- 
tacks of dilatation and palpitation, and with a probable congenital weakness 
of the kidney (37.5 per cent, having a movable kidney). The adults are 
neurasthenics with distinct vasomotor paralysis. 

A type of albuminuria has been observed in some patients with enlarged 
spleen, in which albumin is present when the patient is flat on his back but is 
absent when he is erect. This has been called " hypostatic albuminuria J' Its 
pathogenesis is uncertain, but it can have only an indirect relation to the en- 
larged spleen, as many cases of splenic tumor do not show this albuminuria. 

A still further type of functional albuminuria is known as "albuminuria 
of adolescence.'^ This occurs between the ages of 14 and 16, and then 
disappears. It is different from the cyclic type, although this latter occurs in 
young people. The children are usually anemic, have a neurotic family his- 
tory, have^an unstable vasomotor system and, possibly, a congenital weakness 
of the kidney. In this class should be included the albuminuria shown by 
masturbating children or after sexual excess at this period. The kidney does 
not keep pace with the physical growth and activity of the system, so that the 
association with the unstable vasomotor system may account for the albumin- 
uria (Emerson) . According to Sutherland, movable kidney may be accountable 
for some of these cases, as he finds it present in one- third of his cases. 

Febrile Albuminuria. 

During the course of any acute fever, an albuminuria may be observed 
which is not associated with distinct changes in the renal parenchyma and dis- 
appears with the fall of temperature. The amount of albumin excreted may 
be small or great, depending upon the severity of the toxic action of the bac- 
terial products.^ In ordinary cases there is practically no inflammatory con- 
dition present in the kidney, the albuminuria being due to an ischemia and a 
later hyperemia. In some of the infectious fevers the influence of the toxins 
is so great that a true nephritis originates, as especially noted in scarlet fever 
and diphtheria. Any febrile albuminuria may pass into a true nephritis, so 

1 See Grafe, Deutsch. Arch. f. klin. Med., 1914, CXVI, 328; Lund, Ugesk. f. Laeger, 
i9i5,LXXVII, 1707. 



THE URINE 279 

that the case must be closely watched for the appearance of symptoms indi- 
cating such a complication. In some cases, an increase in the albuminuria is 
observed during convalescence, when only traces were previously noted. 
This is known as ^^colliquative albuniinuria." 
Traumatic Albuminuria. 

A transitory albuminuria may be observed following injuries to the 
kidneys or even after bimanual palpation of this organ. The albumin and 
casts may persist for a variable period with no other signs of renal involve- 
ment. In cases of movable kidney, especially during Dietl's crises, an 
albuminuria may be noted due to obstruction in the renal circulation. In 
cases of ureteral stenosis, an albuminuria may be observed as the result of 
the impeded outflow of urine. This same type of albuminuria may be seen 
after blocking of the ureter by a calculus, pressure from a tumor, or twisting 
of the ureter.^ 

Hematogenous Albuminuria. 

By this type of albuminuria we have in mind one in which the albumin 
is excreted as a result of some alteration in the quality and quantity of the 
normal protein of the blood. On the other hand a distinctly hematogenous 
albuminuria may be the result of the excretion of an abnormal protein. This 
type is observed in purpura, scurvy, pernicious anemia, chronic lead or 
mercury poisoning, syphilis, leukemia, jaundice, cachexia, after the inhalation 
of anesthetics, and in diabetes. 
Toxic Albuminuria. 

This type is directly referable to the influence of various toxic agents 
upon the kidneys. The changes in the kidney may be either of a degenera- 
tive order, leading to a distinct nephritis, or may be purely circulatory. 
Among the substances causing such an albuminuria we find ether, chlorp- 
form, mustard, cantharides, mercury, lead, arsenic and antimony compounds, 
oil of turpentine, potassium nitrate and chlorate, phosphorus, carbolic acid, 
salicyUc acid, tar compounds (aniHne derivatives), petroleum and urotropin.^ 
Neurotic Albimiinuria. 

A sUght transitory albuminuria may be observed in epilepsy (in which 
condition it may not always be found but is present invariably when marked 
cyanosis is seen during the attack), in apoplexy, tetanus, progressive paralysis^ 
exophthalmic goiter, mania, delirium tremens, migraine, brain tumor, injuries 
to the head especially affecting the floor of the fourth ventricle, neurasthenia^ 
and various psychoses. Not infrequently we find neurotic patients showing 
an albuminuria as the direct result of perverted metabolism and not as the 
consequence of pathologic changes in the nervous system. 
Albumintuia with Definite Renal Lesions. 

In acute nephritis an intense albuminuria is a constant and important 
symptom. The more acute the case the larger will be the amount of albumin^ 

1 See Evans, Wynne and Whipple (Bull. Johns Hopkins Hosp., 1912, XXIII, 311), wha 
have shown that a reflex albuminuria results from irritation of the urinary bladder. 

* See Cuntz, Miinch. med. Wchnschr., 1913, LX, 1656; Hydrick, Jour. Biol. Chem.,. 
1914, XVII, p. XXXVI; Scott and Hanzlik, Jour. A. M. A., 1916, LXVII, 1838. 



2 8o DIAGNOSTIC METHODS 

the elimination being generally proportionate to the severity of the disease, 
although some acute cases may show no albuminuria (Herringham^). The 
percentage of albumin varies inversely as the amount of urine, as a rule, so 
that it is much better to excrete a larger amount of urine with a low per- 
centage of albumin than a diminished amount of urine with an increased 
percentage of albumin. The absolute quantity of albumin excreted varies 
from 0.2 to I per cent. It may reach as high as 5 per cent, or higher, in one 
case of Senator being 8 per cent., but this is rare. The total excretion in 24 
hours is rarely over 25 grams. Nephritis of syphiHtic origin appears to be 
associated with the largest outputs of albumin. 

In cases of active renal congestion from exposure to cold or through the 
action of drugs, or in chronic passive congestion due to cardiac, pulmonary, or 
hepatic lesions an albuminuria maybe observed without any trace of an active 
renal lesion. The albumin, in these cases, is small in amount and runs parallel 
to the quantity of urine, thus dijffering from the excretion in true nephritis. 

In chronic parenchymatous nephritis the elimination may be relatively 
large, exceeding, in some cases, that of the acute form.^ In the chronic inter- 
stitial type the albuminuria is very slight, rarely amounting to more than 5 
grams. In this type of nephritis the albumin may be absent at various ex- 
aminations, so that frequent investigations of the urine must be made. In 
amyloid kidney, the urine closely resembles that of the interstitial type of 
nephritis, a total absence of albumin being, however, less frequently observed. 
The serum globuUn in this type of kidney disease is relatively more increased 
than in any other type of renal disorder, so that the albumin-globulin quotient 
may be of some importance in diagnosis. 
Tests for Serum Albumin. 

This protein is soluble in distilled water and is coagulated by heat, if the 
solution be acid, at a temperature varying between 56 and 8i°C. The tem- 
perature at which any protein coagulates on heating will depend upon the 
amount of salts present. This protein is precipitated by absolute alcohol 
and by salts of the heavy metals, as well as by the ordinary alkaloidal pre- 
cipitants. It is levo-gyrate, its degree being represented by the following 
formula (a)i> = — 62.6°. It is precipitated by concentrated mineral acids, 
but is dissolved by somewhat large excess. With acetic acid the precipitate 
first formed is readily soluble in a shght excess of the acid. With con- 
centrated alkali serum albumin forms an alkali-albuminate which is less 
soluble in water than is albumin, but which is soluble in an excess of the 
alkali. This fact accounts for the spontaneous precipitation of albumin in 
a concentrated urine which is alkaline in reaction. 

Numerous tests have been given for the detection of albumin in the urine. 
The writer cannot attempt to describe all of these, but must select, therefore, 
those which he has found most useful. 

^ Trans. Clin. Soc. London, 1901, XXXIV, 34. See Parkinson, Brit. Jour. Child. Dis., 
1916, XIII, 138, Hess, Ztschr. f, klin. Med., 1915, LXXXII, 145; Elliott, Am. Jour. Med. 
Sc, 1915, CL, 806; Klotz, Ibid., 827; Frothingham, Ibid., 1916, CLI, 72. 

* See von Hosslin, Deutsch. Arch. f. klin. Med., 1912, CV, 147; Cook, Jour. Am. Med. 
Assn., 1914, LXII, 684; Williams, 111. Med. Jour., 1915, XXVIII, 186; Macris, Grecemed. 
1915, XVII, 17. 



THE URINE 281 

Before any test for albumin may be made, the urine must be absolutely 
clear. It is advisable always to use fresh specimens, but if these are not at 
hand methods must be adopted to clear up the urine. In the majority of 
cases filtration through several folds of filter-paper will usually accomplish 
this. If this does not succeed, as it practically never does if the urine be 
cloudy from the presence of bacteria, recourse must be had to percipitating 
agents which will carry down the suspension of bacteria.^ Such agents 
are powdered magnesium oxid or carbonate, silicic acid, or saw-dust. These 
substances are thoroughly mixed with the urine and the mixture then filtered 
through double folds of filter-paper or plugs of asbestos fiber. The addition 
of lead acetate or any of the salts of the heavy metals is inadvisable, as the 
precipitates formed will include a large part of the albumin, the other precipi- 
tants not affecting the albumin directly. Occasionally the urine may be 
cleared by centrifugation. 

It has been found that the tests for albumin are rendered more distinct 
if the urine be somewhat diluted. Hallauer has shown that the excess of urea 
and phosphates in a concentrated urine interfere to some extent with the 
dehcacy of the reactions. As a rule, a 24-hour specimen is examined or a 
specimen of the urine voided in the morning and that voided at night. The 
variations of the voidings of the different periods of the day are occasionally 
quite marked, the morning specimen frequently showing no albumin while the 
evening specimen may show quite appreciable amounts. 
Heat Test. 

This test is based upon the principle that serum albumin is coagulated 
by heat especially in the presence of acid. One may use either acetic acid or 
nitric acid, but the conditions of this addition are different in each case. If 
acetic acid be added one must be careful lest he add an excess, as the albumin 
precipitate is soluble in a very slight excess of acetic acid; with nitric acid the 
condition is the reverse, care being taken not to add too little else the albumin 
will not be precipitated by the acid. A few drops of dilute acetic acid are all 
that is required while with nitric acid between one-twentieth and one-tenth 
of the volume of the urine must be added (one to two drops of 25 per cent, 
nitric acid per c.c. of urine). 
Technic. 

A test-tube is filled about three-quarters full of the clear neutral or faintly 

acid urine and heated by directing the flame upon the upper portion of the 

tube, the lower portion being held in the hand. If the fluid remains clear 

and the reaction is acid, no albumin is present. If a cloud is noticed it may 

be rendered more distinct by holding the tube against a black back-ground 

when the upper portion will appear more turbid than the lower. This cloud 

may be due to albumin or calcium phosphate, rarely to calcium carbonate. 

To determine which is the cause, acidulate with a few drops of 5 per cent. 

acetic acid. If the urine becomes clear, the precipitate first noticed is calcium 

1 Vaughan (Jour. Lab. and Clin. Med., 1915, 1, 55) calls attention to the erroneous prac- 
tice of heating these bacteria-containing urines with alkali and filtering. As the alkali 
dissolves the bacterial proteins, later tests will show the presence of albumin, which was 
not present in the urine as voided. 



2^2 DIAGNOSTIC METHODS 

phosphate; if it remains turbid and even increases in intensity of turbidity 
the precipitate is albumin; while if due to carbonates an effervescence will be 
observed. It is wise to boil the urine after the addition of each drop of acid, 
so that the danger of getting an excess of acid may be more easily avoided. 
It is to be remembered that, if the protein be very slight in amount, and 
especially if the urine be originally alkaline, the protein will remain in solu- 
tion owing to the formation of acid albumin. If not enough acid is added, 
the precipitate of phosphates may not dissolve, while if too much be added 
the albumin will dissolve. For these reasons it is better to add the acid after 
boihng. In some cases the fresh urine is already too acid to permit of 
coagulation, so that alkali may be added to diminish the acidity. 

The presence of '^nucleo-albumin" may lead to a wrong interpretation in 
this test. This substance is precipitated in the cold by acetic acid and may 
thus be differentiated. The resinous acids, which are excreted in the urine 
after the intake of such drugs as copaiba, cubebs, and benzoin, are not so 
apt to interfere with this test unless a large excess of acetic acid be used, 
which is never admissible. 

If nitric acid be used in place of acetic acid the urine is boiled as above 
and concentrated nitric acid added to a strongly acid reaction. The nitric 
acid should never be added before boiling the urine nor should the urine be 
boiled after the nitric acid is added, as traces of albumin will be dissolved by 
the hot nitric acid. A flocculent precipitate is indicative of albumin. The 
phosphates and carbonates do not confuse in this reaction as they are readily 
dissolved. The ''nucleo-albumin" is also eliminated by this test as it is 
readily soluble in the excess of acid. In this test the urine should be set 
aside and allowed to cool after the boiling is complete, as albumoses, if present, 
will separate out on cooling as a distinct white flocculent precipitate. A 
precipitate of uric acid may also form on cooling, but this is more granular 
and is usually colored, while the albumin precipitate is white unless an 
admixture of blood be present. 

In case the urine be poor in salts, the tests are improved by the addition 
of a saturated solution of sodium chlorid. The salts hinder to a great extent 
the solution of the albumin by the acids. In such cases, therefore, it is wise 
to acidify strongly the urine with acetic acid and then add one-sixth its 
volume of a saturated solution of sodium chlorid as recommended by Purdy.^ 
The urine is now boiled as above when a precipitate on heating will indicate 
albumin. The nucleo-albumin reaction is slight, the albumoses appear only 
on cooling, while the resinous acids may be precipitated but are soluble in 
alcohol, while the albumin is rendered more compact by this reagent. 
Heller's Nitric Acid Test. 

This test is, perhaps, more frequently employed than any other of the 
tests for albumin in the urine. It has a very wide field of usefulness, although 
it is not as delicate as some of those to be mentioned. ^ 

^ Ulrich recommends the overlaying of the acidulated salt solution with urine as a con- 
tact test. Roberts uses a saturated solution of magnesium sulphate. 

2 See Cavazanni, Policlinico, 1914, XXI, 557; Cronquist, Nord. med. Archiv., 1914, 
XL VII, i; Morner, Ibid., 1915, XL VIII, i. 



THE URINE 



283 



A few c.c. of concentrated nitric acid are placed in a test-tube and the 
urine to be tested is allowed to run slowly down the side of the tube in such a 
way as to form a distinct layer of urine above the acid. Some workers advise 
the addition of the acid after the urine, but if this is done it is much better 
practice to allow the nitric acid to flow from a pipet introduced to the bottom 
of the tube. The writer is always accustomed to allow the urine to flow upon 
the nitric acid from a long pipet so that the urine does not perceptibly mix 
with the acid, the tube being held at an angle of 45 degrees. Albumin, if 
present, is precipitated at the zone of contact in the form of a white opaque 
cloud or ring. This precipitate is acid-albumin which is insoluble in a slight 
excess of acid. A red or reddish- violet transparent ring is always obtained 
with normal urine owing to the reaction of the urinary pigments with the 
nitric acid. If the urine contains abnormal coloring matters this colored 
ring may assume various tints. Thus if bile be present a play of colors 
from red to yellow through blue or green takes place, the green being the 





Fig. 81. — Conical test-glass. 



Fig. 82. — Horismascope. 



characteristic coloration; if indican be in excess this colored ring may be 
bluish or even black; while pigments due to drugs will give colors ranging 
from deep red to violet. This colored ring is usually below the white opaque 
ring due to albumin and tends to extend down into the acid instead of up 
into the urine. If much nitrous acid be present in the nitric acid efferves- 
cence may be observed to such an extent that the ring of albumin may be 
lost. This albumin ring is usually sharply defined and separated both from 
the urine and acid as a white opaque ring, whose breadth will depend upon 
the amount of albumin in the urine. If the tube be allowed to stand for some 
time the ring will lose its distinct outline, a more or less diffuse cloudiness 
rising throughout the urine. 

Albumin is, however, not the only substance to be precipitated by nitric 
acid under the conditions of this test. Thus we find globulin, albumoses, and 
resins precipitated exactly at the line of contact of urine and acids. If the 
urine be heated the albumoses will dissolve while the albumin becomes more 
compact. If this precipitate be due to resins the precipitate will dissolve 
in alcohol or ether while the albumin will remain unchanged. It is sometimes 



284 DIAGNOSTIC METHODS 

wise to shake out the urine with ether before applying this test. The globulin 
ring may be differentiated from that of albumin only by separating these 
proteins by the method to be discussed under Serum Globulin. Globulin is 
usually associated with albumin, is practically never found by itself, and has 
practically the same clinical significance, so that for clinical purposes the 
differentiation of these two bodies is unnecessary. Weinberger^ has re- 
cently shown that the addition of thymol, as a preservative of the urine, 
leads to the formation of a grayish-white ring just at the junction of the nitric 
acid and urine when this test is applied. " Below the ring there is a greenish 
zone extending somewhat into the acid, above it a reddish somewhat smaller 
zone." If this substance is suspected, the urine should be extracted by 
agitation with an equal volume of petrolic ether. A somewhat similar reac- 
tion has been reported by Kenney^ in cases in which several drops of formalin 
were added as a preservative to a small amount of urine. 

Besides these rings at the zone of contact, a further white or yellowish 
ring may .be observed at this point. This ring is found in urines which are 
especially rich in urea and appears as a distinctly crystalline ring due to the 
formation of urea nitrate. If the urine be previously diluted this ring does not 
appear. If an excess of uric acid be present in the urine, we observe, on 
allowing the tubfe to stand for a few minutes, a distinct white ring in the urine 
about I to 2 cm, above the point of contact of the acid and urine. 

If the ntucin-like bodies previously discussed are present in slight excess, 
a diffuse cloud appears throughout the urine if the fluids have been slightly 
shaken or a distinct ring i to 2 cm. above the albumin ring may be observed 
if the urine is carefully added to the acid. This ring is seen in practically 
all urines, is never at the point of contact, and does not appear to have any 
clinical sigiiifitance. 

Instead of perforrning this test in a test-tube as described above, it may 
be done in a conical wine-glass, as recommended by Simon and Ogden, or may 
be employed as recommended by Boston. This latter worker uses a flat- 
pointed pipet into which is drawn from i to 2 inches of the urine to be tested. 
The exterior of the tube is then wiped perfectly dry and the pipet, with its 
upper end closed with the finger, is introduced to the bottom of a bottle con- 
taining pure nitric acid. By lessening the pressure of the finger the acid 
gradually flows up into t' e pipet forming a distinct line of contact between 
it and the urine. The same points mentioned above obtain with this test. 
This modification is very simple and may be recommended for general use. 
A further method of performing this test is the use of the horismascope (see 
cut). The urine is placed in the larger tube C and the nitric acid allowed to 
flow through the capillary tube A so that a distinct line of contact is observed. 
The use of this instrument frequently brings out much more clearly the 
albumin ring than do the other modifications. . 

This test is frequently employed in combination with heat. Two methods 
are available, either one of which may be used, although the results are some- 

* Jour. Am. Med. Assn., 1909, LII, 13 10. 
• 2 New York Med. Jour., 1904, LXXX, 403. 



THE URINE 285 

what different in the two cases. Some workers advise heating the nitric acid 
previous to the addition of the urine. This does not seem wise to the writer, 
as traces of the acid albumin are undoubtedly dissolved by the hot acid and 
may thus escape detection. The writer is accustomed to heat only the upper 
portion of the tube which contains the somewhat diluted urine. In this way 
the urates and albumoses are thrown out of the field of action while there is 
much less danger of traces of albumin being dissolved. If the various points 
included by this test are remembered and each interfering substance removed 
by proper differentiation, this test is practically the most useful and reliable 
one for the detection of albumin in the urine. 
Potassium Ferrocyanid Test. 

A few c.c. of urine are strongly acidified with acetic acid and a few drops 
of a 10 per cent, solution of potassium ferrocyanid are added drop by drop. 
In the presence of albumin a faint turbidity or a flocculent precipitate will 
be observed, depending upon the amount of albumin present. The slow 
addition of the ferrocyanid is necessary, as an excess of this reagent will dis- 
solve the precipitate first formed. In this test albumoses and nucleo-albu- 
min are both precipitated. The former is dissolved on heating the mixture, 
while the latter is detected by the precipitate on the addition of the acetic acid 
unless the acid be added in excess. The urates do not interfere if the urine 
is diluted previous to making the test. 

This test is recommended by many as a much more delicate one for albu- 
min than those previously described. It reacts with a smaller amount of 
albumin, but it does not, in the writer's opinion, give as much general infor- 
mation about the urine as does Heller's test.^ 
Sulpho-salicylic Acid Test. 

This substance may be used either in the form of a 20 per cent, solution 
or in the solid state. If the solution be used it is added to the acidified urine 
in such a way that a distinct hne of contact is formed. Albumin will be 
shown by a distinct white ring at the point of contact. It is preferable, how- 
ever, to add to the urine a small fragment of this substance, when in the pres- 
ence of albumin a turbidity or a white flocculent precipitate will be observed, 
depending upon the amount of albumin. The albumoses are also precipitated 
by this reagent, but dissolve on heating. Neither uric acid nor resins react with 
this substance, while the mucin-like substances are not appreciably affected. 

The test, known as Roch's test, is, perhaps, the most convenient one for 
the use of the general practitioner, as the substance may be readily carried 
in the medicine case and added to the urine at the bedside. ^ 
Spiegler's Test. 

As the original reagent of Spiegler was found to be of little value in many 
cases, JoUes^ has modified it with the following composition: 10 grams of mer- 
curic chlorid, 20 grams of citric acid, and 20 grams of sodium chlorid dis- 
solved in 500 c.c. of water. 

^ See Hektoen and Retinger. Trans., Chic. Path, Soc, 1919, XI, 57. 
^Folin and Denis (Jour. Biol. Chem., 1914, XVIII, 273) advocate this substance as a 
quantitative precipitant of albumin. 

^Ztschr. f. physiol. Chem. 191 2, LXXXI, 205. 



286 DIAGNOSTIC METHODS 

The urine is acidified with acetic acid to precipitate the nucleo-albumin if 
present. This substance is filtered off and the filtrate superimposed by 
means of a pipet upon a few c.c. of the above reagent. In the presence of al- 
bumin a distinct white ring appears at the zone of contact. This reagent 
precipitates the albumoses which are soluble on heating. Nucleo-albumin, 
if present, should be removed before applying the test. In case iodids are 
present, mercuric iodid will be precipitated but inay be removed by 
alcohol. 

This test is the most delicate test for albumin. It shows one part of albu- 
min in 350,000 parts of urine. This is almost too delicate for clinical work as 
it will show albumin in practically every specimen of urine. The writer has 
found this test extremely valuable in cases which showed only a faint reaction 
with Heller's test. If such an urine be treated with Spiegler's reagent a much 
more distinct albumin ring will be observed so that all doubt is thus cleared up. 

Many other tests have been advocated, but the writer does not feel that 
they have any advantages over those outlined above. As far as dehcacy of 
reaction is concerned Spiegler's reagent is the most delicate, the sulphosalicylic 
acid, heat and acid, ferrocyanid, and Heller's test following in the order named. 
It will thus be seen that Heller's test is the least delicate of any of the ones 
spoken of above, but the writer is accustomed to use it in general work, re- 
lying upon Spiegler's reagent to settle mooted points of delicacy of reaction.^ 
As a general working rule it should be said that no one of the less delicate 
tests should be relied upon without being confirmed by some one of the others. 

Quantitative Determination of Albumin. 
Scherer's Method. 

Fifty c.c. of urine are placed in a beaker and heated upon a water-bath. 
Two or three drops of dilute acetic acid are then added and the mixture boiled. 
A flocculent precipitate of albumin should separate out; if not, a drop or two 
more of acid is added until such a precipitate is obtained. The solution is 
then filtered through an ash-free filter which has been previously dried and 
weighed. The filtrate should be tested by Spiegler's reagent to see if any 
albumin has been dissolved. If this shows no albumin, the precipitate may 
then be dried on the filter-paper after being washed with water, alcohol, and 
ether. If the filtrate shows albumin, another test with a fresh 50 c.c. of urine 
must be made. Much time is saved if a larger quantity of urine be originally 
taken, treated as above, and small portions filtered off and tested for albu- 
min. The addition of a small amount of a saturated solution of sodium 
chlorid will facilitate the precipitation of the albumin. The precipitate 
on the filter-paper is dried and weighed, the difference in weight between the 
original dried paper and that with albumin representing the amount of 
albumin in the 50 c.c. of urine used. The total amount is determined by 
a simple calculation. 

^ Leone (Policlinico, 1918, XXV, 224, sez. prat.) has introduced a test which is very deli- 
cate and should prove very reliable and satisfactory for the detection of small amounts of 
albumin. Leone's reaj^ent consists of the following: Potassium bichromate, 10 grams; 
100 drops of 25 per cent, sulphuric acid; 100 drops of glacial acetic acid; and 100 c.c. of 
distilled water. The test is made by the contact method. 



THE URINE 



287 



Esbach's Method. 

This test is carried out in a standard graduated glass tube, known as an 
albuminometer (see cut). This tube is filled with acidified urine to the point 
U and the Esbach reagent added to the mark R. This reagent consists of a 
solution of 10 grams of picric acid and 20 grams of citric acid in one liter of 
distilled water. The tube is now closed with the rubber stopper and inverted 
several times in order to mix thoroughly the contents. It is then allowed to 
stand in a test-tube rack for 24 hours, after which the amount of albumin is 
read off, the graduations on the tube representing the number 
of grams of albumin per liter of urine. 

This test has many fallacies and can give only a very approxi- 
mate determination of albumin. The precipitate does not settle 
evenly. If the albumin reads more than 4 grams per liter, the 
urine must be diluted to a specific gravity between 1006 and 1008 
and must be kept at constant acidity. If the urine contains less 
than J^ gram of albumin per liter the test is absolutely useless. 
Further the room temperature must be kept constant at about 
i5°C. or errors as high as 100 per cent, may occur. Moreover, 
albumoses, uric acid, creatinin, resinous acids, etc., are pre- 
cipitated by this reagent. 

Tsuchiya^s Method. 

This method^ is a modification of the above, the reagent be- 
ing a solution of phosphotungstic acid in acidulated alcohol with 
the following composition: 

Phosphotungstic acid, 1.5 grams 

Concentrated HCl, 5.0 c.c. 

95 per cent, alcohol, q. s. ad., 100. o c.c. 

This reagent is used with the Esbach tube as in the above test 
or in the Purdy centrifuge tube discussed later, giving far more 
accurate results than the Esbach test. Normal urine gives only 
a faint unreadable precipitate, while small amounts of albumin 
as well as large ones are completely precipitated by it.^ This 
albuminous precipitate settles regularly and quickly, foaming or 
floating of the precipitate being rarely seen. Variations in tem- 
perature have little influence on this precipitation. It is to be 
remembered, however, that other urinary albuminous substances ^ f^*^; , ^3--- 

EsD3,cn S 3,1- 

will be also precipitated. Mattice^ has shown that this method buminometer. 
yields fairly accurate results for comparative purposes, the fig- 
ures agreeing closely with those of the gravimetric methods. He has 
also demonstrated that the modification of this test, as introduced by 

iZentralbl. f. inn. Med., 1908, XXIX, 105. 

^ n large amounts of albumin are present, the urine must be diluted, as complete pre- 
cipitation may not occur with the amount of reagent used. The supernatant fluid must be 
water clear before the precipitation can be regarded as complete. 

3 Arch. Int. Med., 1910, V, 313. See, also, Pfeiffer, Berl. khn. Wchnschr., 1913, XLIX, 
114; Semionov, Presse med., 1914, XXII, 579; Kahn and Silberman, New York Med. Jour., 
1914, C, 667. 



206 DIAGNOSTIC METHODS 

Goodman and Stern/ is neither accurate nor satisfactory, largely because 
the point at which albumin is shown by the first turbidity with this reagent 
(and hence the amount of albumin reacting) varies markedly with the 
dilution of the urine. 

The writer advises, therefore, that this method of Tsuchiya be used for 
reliable comparative clinical results, the gravimetric or Kjeldahl methods 
being applied for scientific purposes. In case the latter method be used, 
multiply the nitrogen values of the precipitated and washed albumin by 6.3 
to obtain the albumin values. 

It is perhaps unnecessary to state at this point that in speaking of per 
cent, of albumin, one should have reference only to the number of grams of 
albumin by weight in 100 c.c. of urine. The urine very rarely contains more 
than 5 per cent, of albumin, although Salkowski has reported a case in which 
8 per cent, was observed, the albumin separating out as a white amorphous 
precipitate on standing. This fact should be remembered, as the appearance 
of such a precipitate in the untreated urine might be very misleading. 
Purdy's Centrifugal Method. ' 

To 10 c.c. of the urine placed in a centrifuge tube, 3 c.c. of a 10 per cent, 
solution of potassium ferrocyanid and 2 c.c. of 50 per cent, acetic acid are 
added. The reagents and urine are then mixed by placing the thumb over 
the end of the tube and inverting it several times, after which the tube is 
allowed to stand for 10 minutes. It is then placed in a centrifuge the radius 
of which, with its tubes extended, must be 6^ inches. The tubes are re- 
volved for exactly three minutes at a uniform speed o' 1,500 revolutions per 
minute. The amount of albumin is then read off in bulk percentage, each 
division of the tube representing i per cent., as only 10 c.c. of urine are used 
and the divisions represent tenths of a c.c. One per cent, by bulk represents 
0.021 per cent, by weight of albumin. 

This method is very satisfactory, although not absolutely accurate. It is 
difficult to keep a centrifuge running uniformly at the above rate, unless a 
speed indicator be watched during the entire period. The method is more 
exact and more expeditious than the Esbach method and is to be recom- 
mended, therefore, for clinical estimations of the amount of albumin. ^ 

Removal of Albumin. 

It is advisable in many of the general quantitative tests applied to the 
urine that the albumin should be removed if it is present in more than traces. 
Usually this may be done by acidifying with acetic acid and boihng until the 
precipitate is flocculent. The filtrate in such cases will usually be clear and 
contain no albumin. As this test does not eliminate the albumoses and fur- 
ther as the boihng acid may hydrolyze a small amount of the albumin into 
albumoses, Hofmeister recommends the following method. Ten c.c. of a 40 
per cent, solution of sodium acetate and the same amount of 10 per cent. 

* Jour. Am. Med. Assn., igo8, LI, 2055. Autenrieth and Mink (Miinch. Med. Wchn- 
schr., 1915, LXII, 1417), Claudius (Hospitalstid., 1916, LVIII, 1237) and Marshall, 
Banks and Graves (Arch. Int. Med., 191 6, XVIII, 250) have advanced colorimetric methods 
for the determination of albumin. 

2 See Strzyzowski, Ztschr. f. physiol. Chem., 1913, LXXXVIII, 25. 



THE URINE 259 

ferric chlorid are added to the urine when it will be colored a bright red. The 
urine is neutralized or rendered very faintly acid and is then boiled. The al- 
bumin separates out along with the basic ferric acetate and is filtered off. 
This method is not applicable if glucose is present.^ 

(b) Serum Globulin. 

Serum globulin is associated in the blood with serum albumin. This 
protein is not a single body, but is probably a mixture of two forms known as 
euglobulin and pseudo-globulin. These two fractions differ in their precipita- 
tion and solubility constants and must be looked for in works on physiologic 
chemistry. The former of these probably occurs in most urines, constituting 
a large part of what has been called ''nucleo-albumin" (see above). 

Serum globulin, in the specific sense, is usually found in the urine in every 
case in which serum albumin is observed. Cases are reported in which each 
one of these protein bodies has appeared separately, but this is not the normal 
finding. Its excretion as compared with that of serum albumin varies from 10 
to 75 per cent, of the total protein. The relation of the albumin to the 
globulin of the blood is as 1.5 is to i. This relation, known as the "albumin 
quotient,'' is by no means the same in the urine. In cases of amyloid degen- 
eration of the kidney the globulin may be very much increased beyond that 
observed in other chronic affections of the kidney, the albumin quotient 
being usually lower than one. Senator considers this an important point in 
the diagnosis of this condition. In the various types of nephritis we find the 
globulin being greater the more acute the condition, so that in acute diffuse 
nephritis the albumin quotient may be very low, while in chronic parenchy- 
matous nephritis the quotient ranges from two and five-tenths to five and 
five-tenths. As the nephritis improves the relative amount of globulin 
diminishes, but increases with each acute exacerbation. ^ 

Globulin is insoluble in water, but soluble in dilute solutions of sodium 
chlorid, dilute acids, or alkalies, unless these be exceedingly dilute and their 
action not prolonged. If, therefore, urine containing globulins be highly 
diluted with water the globulin will be precipitated in the form of a distinct 
cloud. The precipitation constants with various inorganic salts must be 
learned from text-books on physiologic chemistry. 
Qualitative Test for Globulin. 

The urine is rendered alkaline by the addition of a few drops of ammonium 
hydrate and the precipitated phosphates filtered off. To the filtrate is then 
added an equal volume of a saturated solution of ammonium sulphate, the 
mixture is allowed to stand one hour and is then filtered. The albumoses and 
nucleo-albumin may also be precipitated in this way. Ammonium urate does 
not usually separate out in the hour. The precipitate on the filter is then 
washed with a half -saturated solution of ammonium sulphate until the filtrate 
is albumin-free. A distinct precipitate is usually evidence of the presence of 

^See Tracy and Welker (Jour. Biol. Chem., 1915, XXII, 55) for the use of aluminium 
hydroxide cream in removing albumin. 

^Belfield (Jour. Am. Med. Assn., 1915, LXIV, 204c) calls attention to the presence of 
globulin, with or without albumin, in conditions associated with leakage of the seminal 
vesicles. 
19 



290 DIAGNOSTIC METHODS 

globulin, as the albumin is not precipitated until the urine is completely satu- 
rated with the ammonium sulphate. In order to eliminate the other factors 
the precipitate is dissolved in water and heated on a water-bath to coagulate 
the proteins. The solution is filtered, the precipitate washed with water and 
heated on a water-bath with a i per cent, solution of sodium carbonate. It is 
then filtered and neutrahzed with acetic acid. If a precipitate occurs it is 
globulin, as the albumoses and nucleo-albumin would not be precipitated by 
such treatment. 

Paton advises the use of a contact method in detecting globulin. The 
phosphates are removed as mentioned above and the filtered urine allowed 
to run down the side of a test-tube containing a few c.c. of a saturated solu- 
tion of sodium sulphate. A white ring will indicate the presence of globulin. 
Quantitative Determination. 

The phosphates are removed as above and 100 c.c. of the clear filtered 
urine are treated with an equal volume of a saturated solution of ammonium 
sulphate or directly saturated with magnesium sulphate. The precipitated 
globulin is collected on a dried and weighed filter and washed with a half- 
saturated solution of ammonium sulphate in the former instance or with a 
saturated solution of magnesium sulphate in the latter case. The final wash- 
ings should show no trace of a reaction for albumin. The funnel with the 
filter-paper and its contents are then dried at iio°C. The ammonium sul- 
phate is washed from the precipitate with hot water and the precipitate is then 
dried with alcohol, ether, and finally at iio°C., until the weight becomes con- 
stant. The difference between the weight of the filter-paper and the filter- 
paper plus the globulin gives the amount of globulin in 100 c.c. of urine. 

(c) Proteoses. 

These are intermediate products of the digestion of protein by ferments, 
by acids, or by bacteria. In the normal digestion we find the products pass- 
ing through the following stages: protein, acid albumin, primary proteoses (of 
which there are two, namely, protalbumose and heteroalbumose), secondary 
proteoses (the only well-established representative being deuteroalbumose), 
peptone, amino-acids and hexone bases. The proteoses and peptones are 
very soluble diffusible bodies which are not coagulated by heating. The 
primary proteoses are precipitated by half-saturation with ammonium sul- 
phate, the secondary proteoses are precipitated only after complete saturation 
with this salt, while the peptones are not precipitable in either one of these 
ways. The primary proteoses are precipitated by nitric acid, thus differing 
from the secondary types which are not so precipitated. In the urine we find 
representatives of both types of proteoses, while more or less doubt exists as 
to whether true peptone has ever been isolated from the urine. 
Primary Proteoses. 
Bence-Jones' Protein. 

This body was first discovered by Bence-Jones^ and was regarded as a 
heteroalbumose. Later work by Magnus-Levy 2 has shown that it is in all 

1 Med. and Chir. Trans., 1850, XXXIII; Phil. Trans. Royal Soc, 1848, 1, 55. 
^Ztschr. f. physiol. Chem., 1900, XXX, 200. 



THE URINE 291 

probability a true albumin, as its digestion products include protalbumose 
which could hardly be derived from true heteroalbumose. As the exact 
chemical nature of this body has not been definitely settled, it will be dis- 
cussed under the above heading, although it probably does not properly be- 
long there. This body differs from all other types of protein material which 
occur in the urine in its property of precipitating when heated to as low a tem- 
perature as 4o°C. and of practically completely dissolving on boiling, to ap- 
pear again on cooling. A second characteristic of this body is the readiness 
with which it dissolves in dilute ammonia after it has been precipitated with 
alcohol. The excretion of this body in the urine has been called ''heteroal- 
bumosuria," "myelopathic albumosuria of Bradshaw," ''Kahler's disease" 
and ''Bence- Jones' albumosuria." 

The amount of this protein body excreted is somew^hat variable. In 
Bence- Jones' original case an output of 6.7 per cent, or a total amount of 70 
grams in the 24 hours was observed, while Coriat^ reports a case in which none 
was found in the urine, although 4 per cent, was present in the pleuritic effu- 
sion. Between these limits we find the majority of cases showing an output 
usually not over i per cent. The literature contains about 35 cases showing 
the excretion of this body. The output of this body appears to be constant 
during the day and not affected in any way by the diet. Little is known re- 
garding the direct origin of this body.^ It undoubtedly has some associa- 
tion with the bone-marrow, but just what is not clear. "We may imagine, 
however, that through the agency of the cells of the abnormal tissue, that is 
their products of metabohsm, the normal transformation of the ingested 
albumin into tissue-albumin is impeded, resulting in the production of 
the substance in question, which is then eliminated as foreign matter" 
(Simon). 

This body is excreted in cases associated with the occurrence of multiple- 
myelomata of the bones, especially when these affect the thoracic skeleton. 
It has been observed in a very few cases of leukemia and in a case associated 
with metastatic carcinoma.^ However, these findings are so rare that the 
presence of this urinary protein may be regarded as practically pathogno- 
monic of multiple myelomata. In some cases of this disease the urine does 
not show the Bence- Jones' body, so that a negative finding does not neces- 
sarily preclude the condition. Ellinger has shown that this disease may take 
its course without the occurrence of local bone symptoms, but may be asso- 
ciated with a marked anemia. It is, therefore, wise in cases of obscure 
anemia to test the urine repeatedly for this body. 

^ Am. Jour. Med. Sc, 1903, CXXVI, 631. 

2Rosenbloom (Arch. Int. Med., 1912, IX, 236 and 255) advances the possibility of osseo- 
albumoid as a precursor of this protein. See, also, Hopkins and Savory, Jour. Physiol., 

1911, XLII, 196; Cathcart and Henderson, Jour. Path, and Bacteriol.. 1912, XVII, 238. 

2 Boggs and Guthrie, Am. Jour. Med. Sc, 1912, CXLIV. 803; Bull. Johns Hopkins Hosp., 

1912, XXIII, 353; Ibid., 1913, XXIV, 368; Folin and Denis, Jour. Biol. Chem., 1914, XVIII, 
277; Schiitz, Deutsch. Arch. f. klin. Med., 1914, CXIII, 441; Sexsmith and Klein, Med. 
Record, 1915, LXXXVIII, 600; Schumm and Kimmerle, Ztschr. f. physiol. Chem., 1914, 
XCII, i; Martiri, Policlinico, 191 s, XXII, 451; Groat and Brewer, Jour. Lab. and Clin. 
Med., 1916, I, 895; Taylor and :\liller, Jour. Biol. Chem., 1916, XXV, 281; Taylor, Miller 
and Sweet, Ibid., 191 7, XXIX, 425; Hermann and Wilson. Lancet. 1918, II, 879; Abder- 
halden, Ztschr. f. physiol. Chem., 1919, CVI, 130. 



292 DIAGNOSTIC METHODS 

Tests for Bence-Jones* Body. 

The specific reaction for this protein is observed on heating the acidified 
urine very slowly. At a temperature varying from 50° to 60° a slight cloud 
changing to a marked turbidity and then into a dense cloud will be observed. 
This may be so intense that the urine appears distinctly milky. This tur- 
bidity may change into a heavy sticky precipitate or coagulum as the tem- 
perature approaches the boiling-point. When the boiling-point is reached 
the precipitate entirely or partially dissolves, especially if the boiling be 
continued from one to three minutes. The precipitate may not absolutely 
all dissolve on boiling, as variations in the acidity of the urine and the amount 
of mineral salts present may affect this process. If the tube be allowed to 
stand after being boiled the precipitate returns as the fluid cools. None of 
the other protein bodies give this sequence of precipitation, dissolving, and 
reprecipitation. Hugounenq suggests the name '^thermolytic albuminuria" 
for the excretion of this body in the urine. 

Upon the addition of concentrated nitric acid, drop by drop, a temporary 
turbidity develops which disappears on shaking, but persists if more acid be 
added. If the mixture be heated the precipitate will dissolve and reappear 
on cooUng. This same reaction may be observed on applying any of the 
tests for serum-albumin outlined above. 

This protein is precipitated from its solution by the addition of two 
volumes of a saturated solution of sodium chlorid to urine which has been 
previously acidified with acetic acid. The addition of two volumes of satu- 
rated solution of ammonium sulphate hkewise causes its complete precipitation. 
It may be then washed with alcohol and ether and dried over sulphuric acid. 

Boston^ has proposed the following test for this body. Fifteen to twenty 
c.c. of filtered urine are placed in a test-tube and mixed with an equal volume 
of a saturated solution of sodium chlorid, the tube being shaken to insure a 
thorough mixing of the fluids. Two or three c.c. of a 30 per cent, solution of 
sodium hydrate are added and the mixture vigorously shaken. The upper 
one-fourth of the mixture is then gradually heated to the boihng-point and 
a solution of 10 per cent, lead acetate added drop by drop, the heating being 
continued after each addition. When the drop of lead solution comes in 
contact with the liquid a copious pearly or creamy cloud appears at the sur- 
face, becoming less dense as the boiling-point is neared; and when ebullition 
is prolonged for from one-half to one minute the upper portion of the liquid 
shows shght browning, which deepens to a dull black color. Standing in- 
tensifies the reaction, and if this be prolonged for several hours the black 
precipitate falls through the clear stratum of liquid, collecting in the bottom 
of the tube as a coarsely granular pigment. This reaction is based upon the 
fact that this body contains a large proportion of loosely bound sulphur. 
Lindemann^ finds that this body really contains no more such sulphur than 
does serum albumin, while Wood could obtain no more blackening than with 
other proteins. This test would seem to have Httle value as it is in no way 
distinctive for the Bence- Jones' protein. 

^ Clinical Diagnosis, Philadelphia, 1905. 

2Deutsch. Archiv. f. klin. Med., 1904, LXXXI, 114. 



THE URINE 293 

The quantitative determination of this body may be made by precipita- 
tion with two volumes of saturated solution of ammonium sulphate and the 
washing and drying of this precipitate as previously mentioned. The Tsuchiya 
method is useful as an approximate estimation in the absence of albumin 

Secondary Proteoses. 

Deuteroalbumose is probably the body which has been found in the urine 
in cases in which peptone was reported. This secondary proteose differs in 
its reactions from the primary proteoses and the Bence-Jones' protein. It is 
precipitated only on complete saturation of the urine with ammonium sul- 
phate. In performing this test the urine should be made albumin-free, 
preferably by the Hofmeister method previously discussed. Nucleo-albumin 
may be precipitated by basic lead acetate. Urine containing deuteroalbu- 
mose does not become cloudy on boiling; does not regularly give Heller's test, 
but does react with the ferrocyanid test when the neutral salts are present in 
fairly large quantities, and reacts in the cold with sulpho-salicylic acid and 
with Spiegler's reagent, but the precipitate dissolves on heating to reappear 
again on cooling. This reaction with the latter reagents in the cold may be 
distinguished from that of albumin by boihng the mixture and filtering while 
hot. The albumin remains on the filter while the albumose is present in the 
filtrate, appearing as a distinct precipitate as the solution cools. 

Tests for Albmnoses. 

To a few c.c. of urine, from which albumin and nucleo-albumin have been 
removed as outlined above, add one-fifth its volume of concentrated acetic 
acid. A 10 per cent, solution of phosphotungstic acid is then added, when 
the urine remains clear on standing if albumoses are absent; while a milky 
turbidity is observed in 5 to 10 minutes if these substances are present. If 
this precipitate be filtered off (warming will facilitate the clumping of the 
precipitate), washed with distiUed water and then dissolved on the filter with 
a very dilute solution of sodium hydrate, the solution will have a distinctly 
blue color. This solution is warmed until it becomes clear, more sodium 
hydrate being added if necessary. It is then cooled and the biuret test ap- 
plied by adding a few drops of strong sodium hydrate and a few drops of 
dilute (2 per cent.) copper sulphate solution. On warming this mixture a 
beautiful red color will be observed. Fittipaldi^ advises that 10 c.c. of urine 
be treated with 60 c.c.of absolute alcohol and allowed to stand until the next 
day. The alcohol is carefully poured off, the precipitate is dissolved in the 
least possible amount of 30 per cent. NaOH and this alkaline solution is 
treated with a few drops of a freshly prepared ammoniacal nickel solution. ^ 
In the presence of albumose or peptone an orange-red coloration appears. 
This test seems reliable and does not react with albumin. 

Pang's Method. 

Ten c.c. of urine are heated in a test-tube with 8 gr^ms of finely powdered 
ammonium sulphate until the salt has been dissolved. The mixture is boiled 

^Deutsch. med. Wchnschr., 1911, XXXVII, 1890. Gazz. d. osp., 1911, XXXII, 515. 
^ A 5 per cent, solution of nickel sulphate mixed with an equal volume of ammonia water. 



294 DIAGNOSTIC METHODS 

for a few seconds and is then centrifugalized for one-half to one minute. The 
supernatant fluid is then poured off and the precipitate extracted with 
alcohol to remove urobihn. After pouring off the alcohol the residue is dis- 
solved in a little water, the solution is boiled to remove albumin and filtered. 
The filtrate is shaken out with chloroform to remove any traces of urobilin 
which may have escaped previously. The watery solution is then poured off 
from the chloroform and tested as above for the biuret reaction. 
Clinical Significance. 

Deuteroalbumose may occur in the urine either alone or associated with 
albumin. It is observed in a great variety of conditions, so that distinct 
types of albumosuria may be noted. 

Large accumulations of pus anywhere in the system lead to the excretion 
of deuteroalbumose as a result of the breaking down of the pus cells and the 
later absorption of the hydrolyzed material. This form, known as pyogenic 
albumosuria, is observed in pneumonia during the stage of resolution, in 
gangrenous processes anywhere in the system, in empyema, bronchiectasis, 
abscess formation, and in epidemic cerebrospinal meningitis. In this latter 
condition the differential diagnosis from a tubercular meningitis often rests 
on the appearance of albumose in the urine. 

A hepatogenous form of albumosuria occurs in any condition associated 
with marked disturbance of hepatic function, as for instance in acute yellow 
atrophy, phosphorus poisoning, cirrhosis, carcinoma, and catarrhal jaundice. 
Little is known of the origin of the albumose in this condition. 

An enterogenous type is observed in cases of gastric or intestinal ulcer, 
whether the latter be due to typhoid fever or dysentery, while intestinal tuber- 
culosis is less frequently associated with the appearance of albumose in the 
urine. In these cases the breaking down of the tissues may be responsible 
for increased absorption both of the products of hydrolysis of the tissues and 
of the food. 

An albumosuria of hematogenous origin has been observed in cases of 
scurvy, leukemia, purpura, dermatitis, poisoning with hemolytic agents, preg- 
nancy, especially after the death of the fetus, and in various psychoses, as well 
as in carcinoma affecting any part of the system. The albumosuria in these 
cases is probably referable to the increased lysis of the cells under the influence 
of the exogenous or endogenous toxins. 

A febrile type is observed in practically all fevers, more especially the 
infectious types, such as measles, scarlet fever, diphtheria, acute articular 
rheumatism, smallpox, and mumps. This is referable both to the influence 
of the toxins of the disease in producing increased protein disintegration as 
well as to the associated septic conditions. 

A large number of other conditions are associated with the appearance of 
albumose in the urine. Such a state is due to the breaking down either of 
tissue or of an exudate, and may, therefore, appear in almost any type of dis- 
ease. In some cases albumose may appear in the urine, following the inges- 
tion of a large amount of albumose. This is the digestive or alimentary 
albumosuria and appears to be indicative of an ulcerative condition some- 



THE URINE 295 

where along the intestinal tract. PoUak^ shows that this albumosuria may 
be due, in part at least, to pathological conditions of the kidney, and suggests 
the name renal albumosuria for such cases. 

As previously stated, albumose may be associated in the urine with 
albumin, constituting the mixed albuminuria of Senator. In these cases the 
albumosuria may precede the albuminuria, may alternate with it, or continue 
after it has disappeared. This condition is particularly prominent in cases 
of nephritis, especially of the syphilitic type, and should be watched with 
care. In any case of albumosuria it is necessary to exclude contaminations 
with foreign material, especially with spermatic or prostatic secretions. 

{d) Peptone. 

True peptone rarely if ever appears in the urine. Peptones are the last 
hydrolytic products of protein which give the biuret reaction. This body is 
not precipitated on saturation with ammonium sulphate as are the other 
types of protein material. Ito reports the finding of true peptones in the 
urine in cases of croupous pneumonia, pulmonary tuberculosis, ulcer of the 
stomach, and in women during the puerperal period. In these cases deutero- 
albumose was also present, so that there is a possibility that the peptone 
isolated by Ito was derived from the urinary albumose. Many reports of pep- 
tonuria are found in the literature, but the substance dealt with in practically 
all of these cases was probably some type of albumose and not true peptone. 

(e) Hemoglobin. 

This body is the normal coloring matter of the blood and is to be regarded 
from the chemical standpoint as a chromoprotein. In the normal metabolism 
disintegration of red blood-corpuscles is constantly occurring, but this is not 
sufficient to lead to a hemoglobinemia and a resulting hemoglobinuria. 
These two conditions must go hand in hand, the latter being impossible 
without the former. 

When the destruction of the red cell becomes so extensive that the accu- 
mulation of the blood pigment in the blood-current is so great that the liver 
is unable to convert it into bilirubin, hemoglobinemia and hemoglobinuria 
must result. 2 While the distinct limit of destruction of cells necessary to 
produce this condition is not definitely settled, it may in general be said to 
occur when approximately one-sixtieth of the hemoglobin of the corpuscles 
is set free. The protein really excreted in the urine is not hemoglobin, but 
methemoglobin, so that the term methemoglobinuria would be better 
used, although a direct hemoglobinemia does obtain. 

From what has been said above it is evident that the excretion of this 
protein will inevitably occur after the use of the so-called hemolytic poisons. 
Among these we find ether, chloroform, snake-venom, arseniuretted hydro- 
gen, phosphorus, hydrogen sulphid, toluylendiamin, mushrooms, anilin, lacto- 
phenin, bile salts, chlorates, pyrogallic acid, naphthol, carbolic acid, carbon 
monoxid, and tuberculin. Hemoglobin will appear in the urine in cases of 
poisoning with the above substances only when the hemoglobinemia is ex- 

' Ztschr. f. d. ges. exper. Med., 1914, II, 314. 

2 See Wilbur and Addis, Arch. Int. Med., 1914, XIII, 235; Addis, Ibid., 1915, XV, 4137 



296 DIAGNOSTIC METHODS 

tensive. In mild cases, the liver will be called upon to form increased biliary 
pigment and the urine will, therefore, contain bile pigments instead of blood 
pigments. Likewise, we find a hemoglobinuria following transfusion of the 
blood of animals into man, after severe burns, exposure to cold, in the course 
of any of the specific infectious diseases, and in malaria^ and syphilis. The 
so-called ''black- water fever" is more probably a malarial hematuria than a 
hemoglobinuria. 

A paroxysmal type of hemoglobinuria has been occasionally reported in 
the literature. This occurs in typical paroxysmal forms after exposure to 
cold or exertion, and is often preceded by a typical "infectious" onset, such 
as chill, fever, and malaise, along with pain in the lumbar region. The hemo- 
globin may be excreted for several days and then disappear with no untoward 
symptoms. This condition is very rare and its cause uncertain.^ An epi- 
demic hemoglobinuria occurs at times in the new-born and is associated with 
a distinct hemoglobinemia, jaundice, and cyanosis. 

The urine, in cases of hemoglobinuria, may be clear, but is generally turbid, 
and varies in color from a bright red to almost a black. The turbidity gives 
the appearance of a peculiar smoky or hazy urine. The urine must be ex- 
amined when freshly voided, as blood-corpuscles soon disintegrate in the 
urine, giving it the same appearance as noted in hemoglobinuria. The clinical 
significance of hematuria and hemoglobinuria are much different and should 
not be confounded. If the urine be centrif uged the supernatant fluid will be a 
clear blood-colored liquid and the sediment will show none or very few red cells. 

The tests for the presence of blood pigments must be applied in order to 
differentiate this protein from the other types. Naturally, any specimen of 
urine containing hemoglobin will react to the albumin tests previously given 
so that it may be very difficult to determine whether a true albuminuria is 
coexistent. As a rule, in such conditions there is an associated nephritis so 
that all the findings of this latter condition may obtain. The chemical tests 
indicate the presence of hemoglobin or of any of its derivatives and do not 
differentiate a hemoglobinuria from a hematuria. Microscopic examination 
for the presence of red blood-cells is the only possible way of clearing up such 
a diagnosis. A spectroscopic examination of the urine will differentiate the 
types of blood pigments (see Blood). 

Tests for Hemoglobin and Derivatives. 
Heller's Test. 
A few c.c. of urine are strongly alkalinized with sodium hydrate and 
heated. Either at once or on standing a brownish-red precipitate of the 

^ Urriola (Interstate Med. Jour., 1Q12, XIX, 74) claims that the black urinary pigment of 
the urine is pathognomonic of malaria. 

2 According to Cooke (Am. Jour. Med. Sc, 191 2, CXLIV, 203), syphilis is the most impor- 
tant, if not the only, etiologic factor in paroxysmal hemoglobinuria. See, however, Lind- 
bom, Hygiea, 1913, LXXV, 885; Ztschr. f. klin. Med., 1913, LXXIX, 147; Young, Jour. 
Am. Med. Assn., 19 14, LXII, 356; Widal, Abrami and Brissaud, Sem. Med., 1914, XXXIII, 
613; Porges and Strisower, Deutsch. Arch. f. khn. Med., 1914, CXVII, 13; Dennie and Rob- 
ertson, Arch. Int. Med., 1915, XVI, 205; Daniels.. Nederl. Tijdschr. v. Geneesk., 1915, 
II, 753; Sabroe, Hospitalstid., i9i5,LVlil, 1056; 1056; Gasbarrini, Policlinico, i9i5,XXII, 
1537; Hintze, Deutsche Med. Wchnschr., 1916, XLII, 1186; Johannessen, Norsk Mag. f. 
Laegevidensk., 1917, LXXVIII, 179. 



THE URINE 297 

phosphates and the carbonates of the alkahne-earths is formed, the color 
being due to the hematin carried down by the phosphates if blood is present. 
If the urine contains a large amount of foreign pigments, this red coloration 
may not be easily noted. In this case filter off the precipitate and dissolve it 
in acetic acid, when the solution becomes red if blood pigment is present, the 
color gradually fading upon exposure to air. If this test be controlled by the 
spectroscopic tests for hematin in alkaline solution it becomes quite reliable 
and very delicate. It indicates one part of oxyhemoglobin in 4,000 of urine. 

Donogany's Test. 

Ten c.c. of urine are treated with i c.c. of ammonium sulphid solution 
and I c.c. of pyridin. If blood be present, the urine will assume a more 
or less intense orange color, which may be more evident on looking through 
the test-tube lengthwise. In this case the hemoglobin has been converted 
into hemochromogen, which may be recognized by the spectroscopic test. 
This test is more delicate than the previous, showing one part of blood to 
8,000 of urine. Instead of the above tests, which are more directly applied 
in urine, the guaiac and aloin tests as discussed under Feces may be applied. 

The spectrum of the various blood pigments will be discussed later, so that 
the writer need only refer to the section on Blood for this. If the urine con- 
tains fresh blood the spectrum is that of oxyhemoglobin, while in cases of 
hemoglobinuria or of hematuria of renal origin the spectrum is that of 
methemoglobin. The urine to be tested spectroscopically should be slightly 
acid and perfectly clear. If a large amount of blood pigment be present, the 
spectrum will be much clearer if the urine be diluted. This dilution should not 
be carried too far, otherwise the absorption lines wiU not appear. In testing for 
methemoglobin the spectrum of neutral as well as of alkahne methemoglobin 
should be looked for. 

(/) Fibrin. 

The occurrence of this protein in the urine is very rare. As fibrin is de- 
rived from fibrinogen through the action of .the fibrin ferment, the presence of 
the former body presupposes that the latter two substances have been present 
somewhere along the genitourinary tract. This substance is an elastic, 
grayish, stringy material insoluble in water and alcohol. Chemically, fibrin 
belongs to the group of globulins; it is soluble with difficulty in dilute saline 
solutions, is coagulated by heat, and precipitated either by great dilution with 
water or by saturation with magnesium sulphate. 

This protein may occur in the urine either in the coagulated form or in 
solution. It is found in any condition in which large amounts of blood are 
present in the urine, whether the blood comes from the kidneys or points be- 
low. It may coagulate immediately after voiding or may occur as preformed 
clots which are formed either in severe inflammations of the pelvis of the kid- 
ney, of the ureter, bladder, or urethra. It occurs also in cases of chyluria 
and rarely in direct nephritis. 

In some cases the fibrin is in solution, especially in urines containing no 
blood. This fibrin separates out in the form of a coagulum on standing or 



298 DIAGNOSTIC METHODS 

may change the urine into a distinctly gelatinous mass. This so-called 
'^spontaneously coagulable urine" is seen more frequently in cases of chy- 
luria, but may be observed in rare cases of nephritis. 
Test for Fibrin. 

The clotted material is filtered off from the urine, is thoroughly washed 
with water and boiled in a i per cent, solution of sodium carbonate. On 
cooling, this solution may be tested as outlined under Serum Albumin. 
(2) Carbohydrates. 

Normally the urine contains traces (o.oi to 0.03 per cent.) of carbo- 
hydrates which are incapable of detection by the ordinary clinical tests. 
Besides these true carbohydrates the urine contains other substances which 
react, especially toward copper solutions, as do the monosaccharides. These 
latter reducing bodies are uric acid, creatinin, conjugated glycuronic acids, 
and various pigments, either normal ones excreted in unusual amounts or 
abnormal ones excreted in usual amounts. The total output of the reduc- 
ing bodies of the normal urine varies between 2 and 3 grams in 24 hours, 
while the normal true carbohydrates of the urine vary from 0.2 to i gram 
per diem. » 

(a) Glucose (d-Glucose) (CH2OH— (CH0H)4— CHO). 

The normal blood contains about one part per thousand of glucose. 
Whether this sugar is in the free state or in combination with other molecules, 
as for instance as the so-called jecorin of Drechsel, is at present an unsettled 
question. According to Claude Bernard, sugar will appear in the urine when- 
ever more than three parts per thousand are present in the circulating blood. 
This figure is, in view of the recent work of von Noorden, Stern, and Lief- 
mann, much too high, as the average finding in their cases was 0.85 part per 
thousand. The excretion of sugar in the urine is known as glycosuria and 
presupposes an excess of sugar in the blood (hyperglycemia). In only one 
form of glycosuria do we find absence of this hyperglycemia; that is, in con- 
ditions in which the kidneys become less impervious than normally to the 
sugar circulating in the blood. This type of glycosuria is most frequently 
observed in cases of poisoning with phloridzin and, also, after intravenous in- 
jection of large quantities of physiologic salt solution (the so-called "salt 
glycosuria"), and has led to the assumption of the clinical entity '^ renal dia- 
betes mellitus.'^ The pathology of such a condition is little understood, so that 
we may for the present disregard this type and limit our discussion to the 
glycosuria which inevitably follows a hyperglycemia.^ 
Glycosuria. 

The normal metabolism is such that any excess of carbohydrate food is 

converted, up to a certain point, into glycogen and stored up in the liver. 

^ See de Langen, Nederl. Tijdschr. v. Geneesk., 1913, II, 1454; Ferannini, Riforma Med., 
1914, XXX, 232; Landau, Rev. de Med., 1914, XXXIV, 145; Strouse and Beifeld, Jour. Am. 
Med. Assn., 1914, LXII, 1301; Salomon, Deutsch. med. Wchnschr., 1914, XL, 217; Galambos, 
Ibid., 13C1; Gram., Hospitalstid., 1915, LVIII, 329; Lewis and Mosenthal, Bull. Johns 
Hop. Hosp., 1916, XXVII, 133; Murlin and Niles, Am. Jour. Med. Sc, 1917, CLIII, 79; 
Beard and Grave, Arch. Int. Med., 1918, XXI, 705; Goto, Ibid., 1918, XXII, 96; Lang- 
stroth. Am. Jour. Med. Sc, 1919, CLVII, 201: Bailey, Ibid., 221; Allen, Wishart and 
Smith, Arch. Int. Med., 1919, XXIV, 523. 



THE URINE 299 

Should this ingestion of carbohydrates exceed the functional power of the liver 
to convert it into glycogen, the excess will pass" through the hepatic filter into 
the circulating blood, thus causing directly a hyperglycemia. Unless in- 
creased muscular activity is sufficient to utilize this excess/ the kidneys will 
excrete sugar until the normal relations again obtain. This is the purely ali- 
mentary type of glycosuria and ceases as soon as the intake is diminished. 
The type of food ingested has much to do with the extent of the glycosuria. 
Under normal conditions it matters relatively little how much carbohydrate is 
ingested in the form of starch, as the products of hydrolysis are gradually ab- 
sorbed and do not lead to overactivity of the liver with a resulting hypergly- 
cemia and glycosuria. On the other hand, a certain limit, different for each 
individual, is observed in the amount of sugar which may be ingested without 
causing a glycosuria. For this reason Naunyn has regarded alimentary gly- 
cosuria as of two distinct types: (i) that following the ingestion of starch, 
which he styles glycosuria ex amylo, and (2) that following the ingestion of an 
excess of sugar, glycosuria e saccharo. The amount of starchy or of saccharine 
food which a person may ingest without a glycosuria is known as the assimila- 
tion limit or degree of tolerance for such food. This factor varies for each indi- 
vidual under normal conditions and under pathologic influences is dependent 
upon the state of the intestines, liver, pancreas, muscles, and kidney. A nor- 
mal person may stand an intake of from 200 to 300 grams of glucose without 
excreting more than traces in the urine, but in the majority of persons this fig- 
ure would, perhaps, be found to be more nearly 150 grams than 300.^ It has 
been found that the administration of as small amounts as 50 grams of galac- 
tose and lactose was followed by an excretion of these sugars in the urine, 
while maltose, dextrose, levulose, and saccharose required much larger intakes. 
The excreted sugar, following the increased ingestion, is in most cases similar 
to that taken in, although Moritz has shown that some of the polysaccharides 
may be partially hydrolyzed into their monosaccharide components. It has 
been found by Worm-Miiller that a large intake of cane-sugar is not followed 
by a maximum excretion of this sugar, even though the assimilation limit has 
been greatly exceeded. Thus he observes, after an intake of 50 grams of 
cane-sugar, an excretion of o.i gram, while after an intake of 150 grams the 
excretion was only 0.85 gram. 

"Alimentary glycosuria occurs in a healthy person only by saturating 
the organism with soluble carbohydrates. Therefore it is absent after admin- 
istering starch, as in this case no more sugar will be absorbed than can be 

^ It must be remembered, however, that a certain portion of this sugar excess is reduced 
to fat and stored up in the tissues. Peirce and Keith (Proc. Soc. Exper. Biol, and Med., 
1915, XII, 210) believe that the kidne}- normally oxidizes a certain amount of glucose and 
that sugar normally gains entrance into the kidney cells in proportion to its concentration 
in the blood, the amount oxidized in these cells depending on the sugar concentration. 

- Woodyatt, Sansum and Wilder (Jour. A. M. A., 1915, LXY, 2067) show that the 
absorption of sugar, following its oral administration, depends on such varied conditions 
that an accurate estimation of the sugar tolerance may be made best by intravenous in- 
jections of sugar. See, also, Blatherwick and Hawk, Jour. x\m. Chem. Soc, 1914, XXXVI, 
152; Macleod, Jour. Lab. and Clin. Med., 1916, II, 112; Taylor and Hulton, Jour. Biol. 
Chem., 1916, XXV, 173; Wilder and Sansum, Arch. Int. Med., 191 7, XIX, 311; Bailey, 
Ibid., 1919, XXIII, 455; Lueders, Ibid., XXIV, 432; Hamman, Can. Med. Assoc. Jour., 
1919, IX, 961; Rohdenburg, Bernhard and Krehbiel, Jour. i\. M. A., 1919, LXXII, 1528. 



300 DIAGNOSTIC METHODS 

metabolized in the body. It is also scanty, or quite absent, if sugar solutions 
be given on a full instead of on an empty stomach. Naunyn has observed, 
regarding alimentary glycosuria, that there is excreted in the urine only that 
sugar which, according to Ginsberg, reaches the general circulation through 
the thoracic duct, thus avoiding the liver. Still other external influences may 
come into play, such as altered capacity of the tissues, especially those of the 
importantly concerned liver. The result of this may be that excessive doses 
of glucose are stored up as glycogen or fat in a given time. For saccharosuria 
and lactosuria the relationships are somewhat different. Here it is very 
obvious that these double sugars, if given in excessive quantities, are not com- 
pletely split up in the intestine or during their passage through the intestinal 
wall, but enter the general circulation as such. The organism, like most of 
the yeasts, cannot decompose these sugars to any extent, so that they leave 
the body with the molecules unaffected" (Magnus-Levy). 

In testing for the pathologic type of alimentary glycosuria, it is customary 
to give ICO grams of cane-sugar or glucose either in the morning on an empty 
stomach or 2 hours after a very light breakfast. The urine of healthy 
persons should remain free from sugar, while a pathologically lowered limit 
of tolerance will be observed by a more or less extensive glycosuria beginning 
in about i hour, reaching a maximum within 2 to 4 hours, and lasting about 
8 hours. If this glycosuria follows the administration of starchy foods, the 
condition is probably a pure diabetic one, while a glycosuria following 100 
grams of grape-sugar does not necessarily indicate a diabetes. It is probable 
that cases showing a somewhat low assimilation limit for sugar belong to 
the type of mild diabetes, although many class them in the general category 
of "hepatic insufficiency." That the liver is incapable in such conditions of 
polymerizing the sugar into glycogen cannot be disputed, but the question at 
issue is, as von Noorden shows, upon what does the insufficiency of the liver 
depend? I quote from the article in his Handbuch der Pathologic des Stoff- 
wechsels as follows: "The cause cannot be overfilHng of the glycogen reser- 
voir, by which we explain the alimentary glycosuria of the healthy. In most 
cases the subjects are in ill health and their previous diet anything but exces- 
sive, so that there is little reason to suppose that their glycogen repository 
was already filled to overflowing. There is no more reason for supposing that 
they have a diminished power of utilizing sugar. ^ In none of the affections 
in question are the processes of oxidation and of energy production decreased; 
rather is there a great increase in oxidation and especially in the combustion 
of carbohydrate in certain of the diseases, such as Graves' disease and high 
fever, which predispose to alimentary glycosuria. Either the liver cells are 
unable to polymerize all the sugar reaching them or else the glycogen formed 
is too quickly converted into sugar, through some increase in the diastatic 
process. The latter would imply that the automatic regulation between 
sugar combustion and sugar formation is no longer so evenly balanced as in 
health. Physiologically the diastatic process is dependent only on the sugar 

1 Minkowski, Lusk, Murlin and, more recently. Roily and David assert that this is not 
true, but that the primary factor in human diabetes is an inadequate utilization of carbo- 
hydrates rather than the overproduction of sugar as von Noorden's theory would demand. 



I 



THE URINE 301 

requirements of the body; here it would also be controlled by the sugar 
supply." 

The idea of hepatic insufficiency as a distinct clinical entity responsible 
for the alimentary glycosuria can hardly hold. One should not be satisfied 
with a mere statement that such a condition exists, but should attempt to ex- 
plain why it does obtain. In many conditions of the liver in which an un- 
doubted insufficiency is present, no glycosuria can be caused by administra- 
tion of a fairly large amount of glucose. On the other hand, administration of 
levulose in these conditions produces a distinct alimentary levulosuria. To 
the classes of cases which give this levulosuria should be, according to Strauss, 
applied the term ''hepatic insufficiency" rather than to those showing an ali- 
mentary glycosuria. . (See p. s^^-) 

It would lead me too far afield to discuss the various factors which influ- 
ence the appearance of sugar in the urine. ^ In many cases a restriction of the 
diet within the assimilation limit will rid the urine of sugar, while in others a 
constant production of sugar within the system occurs. This endogenous 
production of sugar may again be of several types. Thus we find in nervous 
conditions, especially in affections in the region of the fourth ventricle, an ex- 
cretion of sugar which continues only so long as the glycogen of the body is 
not used up. If no carbohydrates be taken in the food, glycosuria will soon 
disappear. This condition is due to interference with the normal nervous 
control of the glycogenic function of the liver. This type of glycosuria, to 
which the name " neurohepatogenous glycosuria'^ has been applied, is found in 
a variety of conditions, such as progressive paralysis, multiple sclerosis, cere- 
bral tumors, peripheral neuritis, traumatic neuroses, mania, melancholia, and 
hysteria. In this type we find the administration of carbohydrates in the 
food being followed by a glycosuria, because the diastatic processes are con- 
tinually urged to increased activity, to such an extent that the hepatic artery 
becomes loaded with sugar. In other words, we have, in this type, an in- 
creased rate of saccharification rather than a diminished formation of glycogen. ^ 

In other cases a continuous glycosuria is observed even after complete 
exclusion of carbohydrates from the diet. In this type there is a constant 
formation of carbohydrates from the protein and fat of the food. The liver 
appears to be capable of storing up glycogen in the usual amount, but the 
system in general is practically unable to utilize the sugar brought to it. In 
consequence of this the liver-cells are repeatedly called upon to convert the 
glycogen reserve into glucose, and as a result the blood becomes laden with 
sugar. Even under these circumstances the hyperglycemia would not neces- 

^See Barrenscheen, Biochem. Ztschr., 1913, LVIII, 277; Woodyatt, Jour. Biol. Chem., 
1913, XIV, 38; Greer, Witzemann and Woodyatt, Ibid., 1913, XVI, 455; Ringer, Ibid., 1914, 
XVII, 107; Roily and David, Miinch. med. Wchnschr., 1914, LXI, 169; Macleod, Jour. Am. 
Med. Assn., 1914, LXII, 1222; Sansum and Woodyatt Jour. Biol. Chem., 1914, XVII, 521; 
Greenwald, Ibid., 1914, XVIII, 115; Woodyatt, Ibid., 1915, XX, 129; Moorhouse, Patter- 
son and Stephenson, Biochem. Jour., 1915, IX, 171. 

2 See Novak, Porges and Strisower, Ztschr. f. klin. Med., 1913, LXXVIII, 413; also, Mann, 
Ibid., 488; Labbe andBouchage, Lancet, 1914, I, 13. It is to be remembered that emo- 
tional excitement, which takes the form of pain, fear, rage, or even slight disturbances 
of mental equilibrium, may lead to a transitory glycosuria. See Hammett, Jour. A. M. A., 
1916, LXVI, 1463. 



302 DIAGNOSTIC METHODS 

sarily cause a glycosuria, providing the normal ferments of the blood arising 
from the internal secretion of the pancreas were present.^ This type of glyco- 
suria is in reality typical diabetes mellitus and is so dependent upon such a 
large number of factors that the writer must refer to other works for its discus- 
sion. Recently Pfliiger has shown that a very close relationship exists be- 
tween the nervous influences of the duodenum and pancreas. His work ap- 
parently indicates that what is generally known as pancreatic diabetes is de- 
pendent to some extent upon disturbance o^ the proper correlation between 
the duodenum and pancreas. A peculiarity in the true diabetic glycosuria is 
that other types of sugar, especially levulose, appear to be well tolerated by 
the diabetic individual without leading to a glycosuria. Besides, the admin- 
istration of the primary hydroly tic products of glucose, as for instance gluconic 
and saccharic acids, apparently diminishes an already existing glycosuria. 
This would seem to indicate that the system is primarily unable to bring about 
the initial cleavage of the glucose molecule. As it has been shown that many 
diabetic cases are associated with sclerosis of the islands of Laegerhans in the 
pancreas, this organ is usually regarded as the principal seat of the pathologic 
changes in true diabetes mellitus. On the other hand, many cases which are 
clinically indistinguishable from those of pancreatic diabetes show no lesion 
in this organ postmortem nor any characteristic lesion in other organs.^ 

Occasionally, cases are seen in which a diplomellituria exists. This is 
the contemporaneous or alternate occurrence of diabetic and non-diabetic 
glycosuria in the same individual (Stern^). The clearing up of one type 
usually reveals the other. A transient glycosuria may obtain in conditions in 
which the oxygen supply is reduced, as in suffocation, poisoning with carbon- 
monoxid, curare and amjd nitrite, or after administration of such drugs as 
strychnine, cocaine, caffeine and adrenalin.* Further, a transient glycosuria 
is not infrequently seen following the use of alcohol, especially in the form of 
beer or champagne. A post-anesthetic glycosuria is observed after the use 
of chloroform and ether, probably as a result of the action of these agents in 
stimulating the transformation of glycogen into dextrose (Hawk).^ 

^ See Clark, Jour. Exper. Med., 1916, XXIV, 621. 

-See Homans, Jour. Med. Research, 1914, XXX, 49; Major, Ibid., 1914, XXXI, 313; 
Bensley, New York, Med. Jour. 1915, CI, 523; Kirk. Arch. Int. Med.. 1915, XV, 39. For 
various phases of carbohydrate metabolism see the following: Janne}^ and Blatherwick, 
Jour. Biol. Chem., 1915, XXIII. 77; Geyelin and Du Bois, Jour. A. M. A., 1916, LXVI, 
1532; Epstein and Baehr, Jour. Biol. Chem., 1916, XXIV, i; Sansum and Woodyatt, Ibid., 
327 and 343: Murlin and Sweet. Ibid., 1916, XXVIII, 261; Janney. Arch. Int. Med., 1916, 
XVIII, 584;'Barringer, Am. Joiir. Med. Sc, 1916, CLI, 181; Lusk, Ibid., 1917, CLIII, 40; 
Janney, Ibid., 44; Allen, Ibid., 313; Mackenzie, Arch. Int. Med., 191 7, XIX, 593; Fitz, 
Ibid., XX, 809; McDanell and Underbill, Jour. Biol. Chem., 191 7, XXIX, 227, 233, 245, 
251, 265, and 273; Palmer, Ibid., XXX, 79; Hoagland and Mansfield, Ibid., XXXI, 501; 
Benedict, Osterberg and Neuwirth, Ibid., 1918, XXXIV, 217; Kamimura, Endocrinology, 
1918, II, 330^ Kleiner, Jour. Biol. Chem., 1919, XL, 153. 

•■*Arch. Diagnosis, 1910, III, 236. 

■* See Emerson (Jour. Am. Med. Assn., 191 2, LIX, 2245) for a discussion of glycosuria in 
the insane; also, Herbert, Biochem. Ztschr., 1913, XLVIII, 120; Landau, Ztschr. f. klin. 
Med., 1913, LXXIX, 201; Folin, Denis and Smillie, Jour. Biol. Chem., 1914, XVII, 519; 
Luzzatto, Ztschr. f. exp. Path. u. Ther., 1914, XVI, 18; Loewy and Rosenberg, Biochem. 
Ztschr., 1914, LXI, 189; Eustis, Am. Jour. Med. Sc, 1914, CXLVII, 830; Anders and 
Jameson, Ibid., 1914, CXLVIII, 323; Pozzo, Policlinico, 1915, XXII, 417. 

^ Arch. Int. Med., 191 1, VIII, 39. See also, King, Moyle and Haupt, Jour. Exper. Med., 
1912, XVI, 178; Greenwald, Jour. Biol. Chem., 1913, XVI, 375; Ross and Hawk, Arch. Int. 
Med., 1914, XIV, 779. 



THE URINE 303 

Qualitative Tests for Glucose. 

The qualitative tests for sugar depend for the most part upon the chemical 
structure of its molecule.^ The hexoses belong either to the class of aldehyds 
or ketones, and as such will reduce metallic oxids to lower forms. The CHO 
and CO groups of the aldoses and ketoses, respectively, are the reacting points 
in all of the reduction tests, such as those with copper and bismuth solutions, 
as well as in the tests showing the formation of the characteristic osazones. 
Moreover, these carbohydrates show the peculiarity of fermenting, in the 
presence of yeast, into alcohol, carbonic acid, and other products. This 
fermentation test, especially with the saccharomyces cerevisiae, is given 
only by the sugars haA'ing three or a multiple of three carbon atoms in the 
molecule. Fischer's work has shown that only those sugars may be fer- 
mented by a specific ferment in which the ferment and sugar stand to one 
another in such a relation that a chemical union is possible between them or as 
he expresses it, only when the ferment fits into the sugar molecule like a key in 
a lock. This explains why only certain types of the hexoses will ferment in 
the presence of yeast. 

Before any qualitative or quantitative test may be made for the pres- 
ence of sugar in the urine, albumin must be removed, especially if present 
in more than traces. - 
Trommer's Test. 

To a few c.c. of urine in a test-tube are added one-third its volume of a 
10 per cent, solution of sodium hydrate and then, drop by drop, a 10 per cent, 
solution of copper sulphate. This copper sulphate should be added with con- 
stant shaking until a slight excess of the precipitated cupric hydrate (Cu (OH) 2) 
remains undissolved and is visible, on shaking the tube, as a distinctly green- 
ish-blue flocculent precipitate. The upper layer of the urine is then warmed, 
when a yellow or red precipitate appears in the heated urine if sugar be pres- 
ent. As a rule, it is unnecessary and even unwise to boil the solution, as 
otherwise substances other than sugar may produce the reaction. It is true 
that the reaction is not as sensitive unless the solution be heated to boiling, 
but other substances do not so readily interfere with the reaction at a lower 
temperature. The yellow or red precipitate will gradually form through- 
out the mixture, from above downward, and will finally settle out, leav- 
ing a colorless or yellow fluid above. 

This reaction is due to the reduction of the cupric hydrate, which is 
formed by the action of the sodium hydrate upon the copper sulphate, into 
cuprous hydrate, which becomes dehydrated, on heating, into cuprous oxid. 
If no sugar be present in the urine and other reducing substances are not ex- 

^ See Levene, Jour. Biol. Chem., 1916, XXIV, 59; Hudson and Dale, Jour. Am. Chem. 
Soc, 1917, XXXIX, 320. 

-See Meinertz, Med. Klin., 1913, IX, 1771; Glaserfeld, Med. Klin., 1914, X, 1388, 1413 
and 1452; Tirard, Lancet, 1914, II, 1133; Geelmuyden, Norsk Mag. f. Laegevidensk., 1915, 
LXXVI, 985; Folin (Jour. Biol. Chem., 1915, XXII, 327) and Benedict and Osterberg, 
(Ibid., 1918, XXXIV, 195) have introduced tests which remove practically all the reducing 
substances in urine with exception of sugar, and thus afford opportunity for detecting the 
normal urinary sugar. These tests are, however, too delicate for clinical purposes as they 
show sugar in almost every urine examined. Hiller, Ibid., 1917, XXX, 125, uses a m'odifica- 
tion of Folin's method to determine pathological glycosurias. 



304 DIAGNOSTIC METHODS 

cessive in amount, the cupric hydrate will settle out on warming as a black 
precipitate of cupric oxid. The equations showing these points are as follows : 

CUSO4 + 2NaOH = Na2S04 + Cu(0H)2 
Cu(0H)2 + heat = CuO + H2O 

2CuO + glucose (CHO - (CH0H)4 - CH2OH) = CU2O + gluconic 
acid (COOH - (CH0H)4 - CH2OH). 

Whether a yellow or a red precipitate forms will depend upon the alka- 
linity of the solution, the stronger the alkalinity the more pronounced is the 
red color due to cuprous oxid, while in less strong alkaline solutions the yellow 
color of cuprous hydrate will predominate. Certain substances normally 
present in the urine as well as some which may be added to it have the 
property of holding in solution the cupric hydrate first formed. This 
property is shown by the deep blue color which the solution assumes. This 
color is very intense in the presence of sugar, but it is unwise to assume the 
presence of sugar from this fact alone. Among the substances which dissolve 
cupric hydrate and which may be present in the urine in varying amounts we 
find ammonium compounds, albumin, uric acid, creatinin, allantoin, mucin, 
glucose, lactose, maltose, pyrocatechin, hydroquinon, alkapton acids, bile 
pigments, and glycuronic acid. On warming the solution, which may not 
contain sufficient amounts of glucose to give a typical reaction, a shght reduc- 
tion of the copper solution will occur leading to the formation of a dirty 
yellow solution. If these bodies be present in excess a distinct precipitate 
may occur, and as a result marked confusion may arise regarding the presence 
of sugar. 1 It has been shown that uric acid and creatinin do not readily 
reduce at as low a temperature as does sugar, so that slight warming is much 
better than boiling. The presence of these bodies very frequently leads to a 
change in color from the bright blue to the greenish-yellow which may be due 
to the presence of sugar in small amounts, but should never be regarded as 
indicative of a pathological glycosuria. 

It should be a working rule, therefore, that mere decolorization of the 
fluid should not be regarded as due to sugar. Glucose reduces so much 
more markedly than the other bodies mentioned that a distinct granular pre- 
cipitate either of cuprous oxid or hydrate forms and settles out, leaving a 
supernatant fluid partially or completely decolorized. Normal urine or 
urine containing an excess of uric acid, creatinin, or ammonium compounds 
practically never produces an immediate precipitate unless the solution be 
boiled for some time. This is due to the fact that these substances hold in 
solution the amount of cuprous oxid which is formed by their reducing action. 
If such solutions be allowed to stand for a time a reddish-yellow precipitate 
may occur, but this should not confuse as the typical sugar reaction occurs im- 

^See Schulz, Ztschr. f. physiol. Chem., 1912, LXXVII, 121; also, Salkowski, Ibid., 1912, 
LXXIX, 164. According to the more modern theory of Colloid Chemistry, the reducing 
action, as indicated by the variation in color of the precipitate or the formation of a greenish 
solution, is due to the formation of cuprous oxid of different degrees of subdivision, that is, 
possessed of different degrees of dispersion. Substances tending to hold this cuprous oxid 
in solution, that is, in a state of high dispersion, do not yield the precipitates as readily as 
they form the greenish solutions. See Fischer and Hooker, Jour. Lab. and CHn. Med., 
1918, III, 368. 



THE URINE 305 

mediately on warming unless too much copper solution be added. In case 
only traces of sugar be present, we may obtain little or no positive reaction 
because the sugar holds in solution the traces of cuprous oxid formed just as 
do the other substances above mentioned. As a rule, however, sugar in patho- 
logic amounts shows a reducing action over and beyond its dissolving action, 
so that a red or yellow precipitate must settle out. The limit of this test for 
sugar is about 0.2 per cent., in which case the reduction will occur as in normal 
urine without the separation of the characteristic copper precipitate. Even 
in this case the yellow color is somewhat more intense and is clearer than the 
dirty yellow color produced in the presence of excessive uric acid or creatinin. 
If the urine contain an excess of the conjugated glycuronates, the test 
may be quite as distinctive as that for sugar. To differentiate such reactions, 
one should resort either to the phenylhydrazin or the fermentation tests. 

Benedict's Test. 

This qualitative test^ is one of the most reliable and accurate modifica- 
tions of Trommer's test. The reducing action of glucose in alkaline solution 
is diminished by strong alkalies such as the hydrates. This property may 
prevent such well-known solutions as Fehling's and Haines" (discussed below) 
from demonstrating small quantities of sugar. Further, the urine contains 
many compounds which interfere with the detection of glucose by the strongly 
alkaline solutions, the loss of delicacy in the other tests being probably due to 
the fact that normal reduction of the solution is inhibited for a period long 
enough to allow the strong alkali to decompose the reducing substance. 
However, in solutions of the alkali carbonates the reduction develops more 
slowly and is not destroyed by this weak alkali. The addition of sodium 
citrate, instead of the Rochelle salt as used by Fehling, makes the solution 
very stable, although little more than does the glycerin of Haines' solution. 
For the reasons given, this solution of Benedict is more sensitive (about ten 
times) to urinary sugar than are the other copper solutions. Moreover, 
it is not appreciably reduced by creatinin and uric acid. It is, however, 
promptly reduced by the alkapton acids and the conjugated glycuronic 
as well as by an excess of such preservatives as chloroform, chloral and 
formaldehyd. The formula of the reagent is as follows: 

Copper sulphate (C. P. crystallized), 17.3 grams 
Sodium or potassium citrate, . i73-o grams 

Sodium carbonate (crystallized)," 200.0 grams 

Distilled water, q. s. ad., looo.o c.c. 

1 Jour. Am. Med. Assn., 1911, LVII, 1193. See, also, Kleiner, Ibid., 1914, LXII, 1307; 
Sheaff, Ibid., 191 5, LXV, 11 81; Weinberger, Am. Jour. Med. Sc, 1914, CXLVII, 407; Folin 
and McEllroy (Jour. Biol. Chem., 1918, XXXIII, 513) have introduced a qualitative 
test for sugar in urine, which has the following composition: 100 grams of sodium pyro- 
phosphate, 30 grams of crystallized disodium phosphate and 50 grams of anhydrous 
sodium carbonate are dissolved in about i liter of water. To this solution is added 13 
grams of copper sulphate dissolved in about 200 c.c. of water. The test is used in the same 
way as is Benedict's but a point of distinction is that, unless a very marked turbidity is 
noted in the hot solution, the result must be regarded as negative. 

2 Or one-half the amount of the anhydrous salt may be used. 



3o6 DIAGNOSTIC METHODS 

Dissolve the citrate and carbonate (with aid of heat) in about 700 c.c. of water 
and filter if necessary. Dissolve the CuSOj in about 100 c.c. of water and 
pour into the alkaline solution. Cool and make up to i liter. 

The technic is as follows: 5 c.c. of the reagent are placed in a test-tube and 
not more than 8 or 10 drops of urine are added. The mixture is then heated 
to vigorous boiling for one to two minutes and allowed to cool spontaneously. 
In the presence of glucose, the entire body of the solution will be filled with a 
precipitate which may be red, yellow or greenish in tinge. If the quantity 
of glucose be low (under 0.3 per cent.) the precipitate forms only on cooling. 
If no sugar is present, the solution either remains perfectly clear or shows 
a faint turbidity, which is blue in color and consists of precipitated urates. 
This test may be especially recommended for all qualitative work. 

Fehling's Test. 

This is, perhaps, the best-known test for sugar but it is inconvenient and 
frequently inaccurate for the reasons above mentioned. The formulae for 
the solutions are as follows: 

Solution A. Solution B. 

Copper sulphate, 34-64 gm. Rochelle salt, 1.73 gm. 

Distilled water, q. s., ad., 500 c.c. Sodium hydrate 50 gm. 

Distilled water, q. s., ad., 500 cic. 

In performing the test with Fehling's solution equal parts of solutions A 
and B are taken and the mixture brought to a boil. The urine is then added, 
drop by drop, when a reduction of the copper solution will appear in the 
presence of sugar. ^ The amount of urine added should rarely exceed 10 
drops, at the outside 20, if a reduction is to be taken as typical and the solu- 
tion should not be boiled for more than a few seconds after adding the urine. 
The addition of larger volumes of urine will usually introduce errors from the 
factors mentioned under Trommer's test. This test has the same points of 
interest as has Trommer's test, so that it is wise to dilute the urine before 
making the test. It is more frequently used than is Trommer's test, but it 
is not as convenient. 

Haines' Test. 

Haines has introduced a modification of Trommer's test by adding 
glycerin, instead of Rochelle salt, to increase the amount of copper in solution. 
This test is much more convenient than Fehling's, the solution having the 
advantage of keeping almost indefinitely. It is, however, far less delicate 
than is Benedict's and is reduced more readily by preservatives as well as 
by excess of many normal urinary constituents, especially by uric acid and 
creatinin.2 

VSee Cramer, Biochem. Jour., 1915, IX, 71; Matriri, Rif. Med., 1916, XXXII, 1264; 
Angiolani, Policlinico, 1917, XXIV, 173. 

2 The creatinin may be almost entirely removed from the urine, if desired, by adding to 10 
c.c.^of urine about 2 grams of picric acid and about 2 grams of Merck's blood charcoal, shak- 
ing'[,for a few minutes and filtering (see Folin, Jour. Biol. Chem., 19 15, XXII, 327). See, 
also, Benedict and Osterberg, Jour. Biol. Chem., 1918, XXXIV, 195. 



THE URINE 307 

The composition of Haines' qualitative solution is as follows: 

Copper sulphate, 12 grams 

Potassium hydrate,^ 45 grams 
Glycerin, 90 c.c. 

Water, q. s., ad., 1000 c.c. 

A perfectly clear, transparent, dark-blue liquid results which throws 
down a very slight reddish deposit of cuprous oxid on standing a week or 
more.^ This does not affect the value of the solution, as the clear blue 
solution is simply decanted as required. 

Four or five c.c. of this solution are placed in a test-tube and gently boiled. 
Six drops of the suspected urine are added and the upper portion of the mix- 
ture brought to a boil and immediately removed from the flame. If sugar be 
present an abundant yellow or yellowish-red precipitate is thrown down; 
if no such precipitate occurs sugar is absent. The precautions to be observed 
in using this test are never to add at the outside more than 10 drops of urine 
and not to boil the mixture for more than one or two seconds after the 
addition of urine. 

Haines' Modified Test. 

Haines and Pond,^ with whom the author collaborated in regard to cer- 
tain features of the work, have made certain modifications in the above 
Haines' solution, in order to increase its delicacy and, also, to permit of the 
performance of the sugar test by the ring or contact method. This modifica- 
tion enables one to detect with certainty 0.03 per cent, of sugar, which is 
about the average of the so-called '^ normal" sugar of the urine. In other 
words, the delicacy of the test is increased so that sugar in pathological 
amount will be detected. While this test is not as delicate as the special 
solutions of Folin and of Benedict and Osterberg mentioned on page 303, 
yet the new Haines' solution has the advantage over these latter solutions 
that it will show sugar only in pathological amounts, while the other solu- 
tions mentioned will show sugar in practically every specimen examined. 
It is, therefore, a clinical test. Owing to the increase of the specific gravity 
of the solution, by the addition of the larger amount of glycerin, the employ- 
ment of the contact test becomes a matter of the greatest simplicity. How- 
ever, one precaution must be taken before this test may be applied. The 
phosphates of the urine must be removed before a clear-cut reaction is ob- 
tained, as their presence causes the formation of a white ring at the point of 
contact of the two solutions, which prevents a distinct sugar reaction from 
showing. This removal may be made by adding to the urine in a test-tube 
5 or 6 drops of a 10 per cent, solution of sodium hydrate and allowing the 
phosphates to settle out or centrifuging if desired to hasten the process. It 
was thought that this precipitation of phosphates could be averted by the 

^ Or 32 grams of sodium hydrate. 

^ The glycerin obtainable at present may reduce the solution in a few hours. If the 
solution be allowed to stand in a warm place for 48 hours, the supernatant fluid iliay be 
decanted or filtered and will remain clear for almost an indefinite period. 

3 Jour. A. M. A., 1920, LXXIV, 301. 



308 DIAGNOSTIC METHODS 

addition of citrate or tartrate to the copper solution, but this was found to 
be ineffective. 

The composition of the modified Haines' solution is as follows: 

Copper sulphate 5 grams 

Glycerin 250 c.c. 

Potassium hydrate^ . 20 grams 

Distilled water to 1000 c.c. 

Dissolve the copper sulphate in a mixture of the glycerin and an equal amount 
of water, with the aid of gentle heat. The potassium hydrate should be dis- 
solved in about 200 c.c. of water and added to the copper solution with con- 
stant stirring, the whole being made up to volume with distilled water. 

The above solution may be used in the same manner as directed for the 
original Haines' solution, but a much more delicate and beautiful reaction is 
obtained as follows: Heat about 5 c.c. of the copper solution to boiling in a 
test-tube, remove from the flame, and hold at an angle of 30 to 40°. To this 
add, by means of a medicine dropper, 10 to 20 drops of the urine, freed from 
phosphates as above mentioned, in such a manner that a distinct zone of con- 
tact is formed between the copper solution and the urine. The tube is then 
placed in the upright position and the reaction noted. If sugar is present in 
quantities exceeding o.i per cent., a brick-red or yellowish ring will imme- 
diately appear at the junction of the two liquids. If the amount of sugar be 
less than o.i per cent., ranging down to 0.03 per cent., the ring will appear in 
from a few seconds to slightly less than a minute, the smaller quantities show- 
ing slower reactions with a tendency to a more yellowish color of the ring. 
In urines containing no sugar, no ring of any kind will be noted at the zone 
of contact. 

This test has the following advantages: (i) the reaction is concentrated 
to a single plane, thus increasing its visibility; (2) only one heating is neces- 
sary and no long standing before the reaction appears; (3) it will demonstrate 
pathological sugar in amounts greater than 0.03 per cent.; (4) it gives 
a clear-cut decisive result in a minimum of time. 

Many other modifications of the copper test have been introduced, both 
for qualitative and quantitative purposes, but they do not have any ad- 
vantages over those mentioned especially over Benedict's and Haines' 
tests. These are used daily in the writer's laboratory and have been found 
very reliable and serviceable. When other tests are used an error may 
creep into the report which can be corrected only by resort to fermen- 
tation and other confirmatory methods. 

Almen-Nylander^s Test. 

This test is a modification of the original Bottger test and is a distinct 
improvement. The reagent is prepared as follows : Four grams of Rochelle 
salt are dissolved in 100 c.c. of 10 per cent, sodium hydrate solution with 
gentle heat, and as much bismuth subnitrate is added as will dissolve (about 
2 grams). After the mixture is cooled the undissolved bismuth subnitrate 

' Or 14.3 grams of sodium hydrate. 



THE URINE 309 

is filtered off and the filtrate kept in a dark bottle, where it will remain 
permanent for a long period. 

To a few c.c. of urine in a test-tube is added one-tenth of the volume of 
this reagent and the mixture boiled for a few minutes. If glucose is present, 
the fluid will darken and a black precipitate of metallic bismuth separate 
out. This black precipitate must occur while the solution is being warmed 
and not after it has cooled. If only a small amount of sugar be present the 
phosphates precipitated by the alkali may be slightly gray in color instead 
of the usual white. This test is somewhat more delicate than is the copper 
test, as it will show about J-^o P^r cent, of sugar. 

This reagent is not reduced by uric acid, creatinin, pyrocatechin, hydro- 
quinon or homogentisic acid; but it is reduced by the conjugated glycuronic 
acid, excess of urinary pigment and pentoses. It is particularly necessary in 
this test that albumin be removed, as bismuth sulphid forms in the presence 
of albumin, and may precipitate either in the form of a reddish or a distinctly 
brownish, even black precipitate. The reduction observed after the adminis- 
tration of medicaments, such as rhubarb, senna, antipyrin, camphor, salicylic 
acid, salol, sulphonal, trional, quinin, eucalyptus, oil of turpentine, and chloral 
hydrate, is usually a brown rather than a black unless the solution stands for 
some time. The administration of saccharin usually results in a reduction of 
this test, while the copper tests are not affected by it. Sulphur bodies 
(methyl mercaptan) excreted after the patient has eaten asparagus give a 
distinct precipitate which may be more or less confusing. If the urine be 
ammoniacal the reaction may not appear in a characteristic way owing to the 
fact that the free ammonia is evolved and the alkalinity of the solution is 
reduced by the combination of the sodium ion with the acid radical formerly 
bound to the ammonium ion. 

This test is used by many as a routine test for sugar, as it is practically 
always negative with normal iirine. The reducing action of the glycuronates 
and pentoses must, however, be remembered. ^ 
Fermentation Test. 

As previously stated, the reduction tests do not absolutely prove the pres- 
ence of sugar. All that one can say is that a reducing substance is present and 
if the reaction be typical the probability is that the reducing substance is 
sugar. The fermentation test is, perhaps, the most certain of all the tests 
for glucose and depends upon the fact that only those sugars which contain 
three or a multiple of three carbon atoms are fermentable with yeast. ^ 
Not all members of these groups of sugars will ferment with yeast, so that 
for absolutely scientific purposes fermentation will not differentiate them. 
Fischer's work along this line should be carefully read by anyone interested 
in the biologic properties of the various types of sugar. As the hexoses, 
which occur in the urine are practically limited to glucose and levulose, we 
are safe in saying that any sugar fermenting with yeast is one or the other of 
these monosaccharides, which may be differentiated by tests to be outlined later. 

^See Mende, Miinch. Med. Wchnschr., 1914, LXI, 11 20. 
^ ^Boros (Med. Klin., 1913, IX, 874) shows that yeast cells may be so numerous in the 
urine, as voided, to destroy the sugar present. This is known as masked or occult glycosuria. 



3IO DIAGNOSTIC METHODS 

The test is performed as follows: Ten c.c. of urine are placed in a test- 
tube and a piece of compressed yeast, which should be perfectly fresh, about 
the size of a pea is added and the urine gently shaken until the yeast is finely 
divided. This mixture is then poured into a fermentation tube, which is 
allowed to stand in the incubator for a few hours. Two control tests, using 
in one normal urine and yeast and in the other normal urine, yeast, and a 
trace of dextrose, are then made and placed in the incubator along with the 
suspected urine. The presence of sugar is indicated by gas (CO2) in the upper 
portion of the fermentation tube. The rapidity of formation of this gas de- 
pends upon the amount of yeast as well as upon the age of the yeast. The 
test indicates from o.i to 0.05 per cent, of sugar, especially if the urine be 
sterihzed by previous boiling. The compressed yeast as usually purchased 
develops a certain minimal amount of gas in normal urine, so that the control 
test is necessary both to show whether ''self -fermentation" is excessive and 
also whether the yeast is at all active in producing CO2 from glucose when it 
has been added. It is necessary, moreover, that decomposition of the urine 
be prevented, either by previously boihng the urine, addition of a trace of 
sodium fluorid, or tartaric acid. 

This test has the great advantage that the other substances, which reduce 
copper and bismuth solutions, do not ferment. If precautions are observed 
to add the right amount of yeast, not to shake the yeast and urine violently 
enough to include much air, and to prevent bacterial decomposition, this 
test will positively show the presence or absence of glucose or levulose in the 
urine and no other substances. It is always wise to use this test either as 
confirmatory or decisive in conjunction with the previous reduction tests. 
Phenylhydrazin Test. 

This test is much more delicate than any of the previous tests mentioned. 
Theoretically it will show sugar in the amount present in normal urine, but 
practically no definite reaction is observed. The principle of the reaction is 
the decomposition which occurs between the aldehyd or ketone group of the 
sugar molecule and the amino group of the phenylhydrazin. In this reac- 
tion, characteristic bodies known as hydrazones are first formed, which are 
converted, in the presence of dilute acetic acid and an excess of phenylhydra- 
zin, into crystalline bodies known as osazones. These latter bodies are char- 
acteristic of the sugar group, usually dififering from one another, depending 
upon the original sugar from which they were formed. The osazones crystal- 
lize in definite forms, the purified crystals showing rather sharp melting points. 
It is, therefore, essential not only that a crystalline body be obtained in this 
reaction, but that the crystalline form and the melting-point of the crystal be 
that characteristic of the sugar suspected. The success of the test will depend 
largely on the relation of the sugar to the reagents, the best proportions being 
approximately one of sugar, two of phenylhydrazin, and three of sodium 
acetate. All of the members of the hexose and pentose groups show this 
reaction, as do man.y of those of the polysaccharide series. As will be seen 
from the reaction given below, those sugars which differ only in the space- 
relations of the atoms attached to the first two carbon atoms can possibly 



PLATE IX. 




OsAZONS. {Hawk.) 
Upper form, dextrosazon; central form maltosazon; lower form, lactosazon. 



THE URINE ' 311 

give the same osazone. For this reason we find glucose, levulose, and man- 
nose as well as glucosamine giving exactly the same osazone (phenylglucosa- 
zone), showing a characteristic yellow needle-shaped crystalline deposit, with 
a melting-point of 204 to 205°C. The reactions leading to the formation of 
this body are shown in the following equations: 

CH2OH - (CH0H)4 - CHO + NH2 - NHC6H5 = CH2OH - 
(CH0H)4 - CH = N - NHC6H5 + H2O 

CH2OH - (CH0H)3 - CHOH - CH = N - NHC6H5 + NH2 - 
NHCeHs -= CH2OH - (CH0H)3 - C - CH -f 2H2O 

II II 

C6H5 - N N - NHC6H5 

Twenty-five ex. of urine are treated with a few drops of a solution of lead 
acetate and filtered to remove the albumin if present. To the filtrate, which 
must be acidified with acetic acid if it is not already acid, is then added 
phenylhydrazin hydrochlorate (3^^ to i gram) and about 2 grams of sodium 
acetate. The tube is then shaken thoroughly to mix the contents and is- 
placed in a boihng water-bath for from one to two hours. Some workers 
recommend a shorter period, such as 20 minutes, but the writer has never 
been able to get as good results with the short heating. At the end of this 
time the material in the tube is filtered while hot and the filtrate allowed to 
cool slowly rather than as some advise to cool suddenly by immersion in cold 
water. If glucose be present a yellow crystalline deposit will appear, which 
under the microscope will show the characteristic needle-shaped crystals 
arranged in bundles or sheaves. This microscopic examination does not 
absolutely prove the crystals to be phenylglucosazone so that a melting- 
point determination must be made before definite proof is forthcoming. For 
this purpose the crystals must be purified by dissolving in a hot 60 per cent, 
alcohol and recrystallizing by adding water and evaporating the alcohoL 
A few of these crystals are then placed in a perfectly dry capillary tube and 
the melting-point determined by methods previously learned in organic 
chemistry. 

Neumann has applied Fischer's method of using this test to the urine. 
Five c.c. of urine are treated in a test-tube with 2 c.c. of a 50 per cent, solution 
of acetic acid saturated with sodium acetate and two drops of pure phenyl- 
hydrazin. This mixture is then evaporated by boiling to 3 c.c, after which 
it is cooled quickly and is then re warmed and allowed to cool slowly. If 
glucose be present to the amount of 0.02 per cent., pure crystals of phenyl- 
glucosazone separate out in 5 to 10 minutes. If the urine has a high specific 
gravity and a low sugar content the crystals do not form so quickly. This 
test is much to be preferred in well-equipped laboratories, but is not as con- 
venient as the use of the crystalUne phenylhydrazin hydrochlorate for the gen- 
eral worker, as the fluid phenylhydrazin is quite irritating and is not as easy 
to work with. 

This test cannot be used for quantitative purposes as the yield is never 
complete. For qualitative purposes, however, it is to be especially recom- 



312 DIAGNOSTIC METHODS 

mended. When properly applied it is the most delicate and one of the most 
reliable tests at our disposal. If the melting-point of the crystals be deter- 
mined the only confusing substances will be levulose, glucosamin, and man- 
nose. The osazones formed from the other carbohydrates and the glycuronic 
acid compounds crystallize in somewhat similar form, but do not show the 
characteristic melting-point of 204° to 205°C. of the phenylglucosazone. It is 
true that the impure crystals melt at somewhat lower temperature, but if care- 
fully purified from 60 per cent, alcohol they will melt at approximately 204°. 

Quantitative Methods. 
Folin and Peck^s Method. 

This method^ is a revision of that of Folin and McEllroy,^ which, in turn, 
is a modification of that of Benedict.^ It is accurate and reliable, if aU 
precautions noted are followed, and can be recommended. 

Reagents Necessary. 

1. A copper sulphate solution, containing 59 grams of CUSO4.5H2O and 
2 c.c. of concentrated sulphuric acid per liter. The acid is added to prevent 
the gradual decomposition of the copper sulphate to copper hydrate or 
silicate, which is presumably caused by the solvent action on the glass. 

2. An approximately saturated sodium carbonate solution (14 to 20 per 
cent. Na2C03). 

3. A salt mLxture containing 200 grams of crystallized disodium phos- 
phate, 50 grams of sodium thiocyanate (or 60 grams of the potassium salt), 
and 100 grams of anhydrous sodium carbonate (or 120 grams of mono- 
hydrated sodium carbonate. As difficulties have been encountered in the 
proper preparation of this mixture, as pointed out by Haskins^ and others, 
the following method should be followed: Powder in a large mortar 200 
grams of the disodium phosphate (Na2HP04.i2H20) and sprinkle over it 
the 50 grams of sodium thiocyanate. Mix for 10 minutes with mortar and 
spoon giving a uniform semi-liquid paste. Add the sodium carbonate and 
mix with mortar and spoon until a rather fluffy granular powder is obtained. 
Test this mixture for proper mixing by adding 5 grams of it to 5 c.c. of the 
above copper sulphate solution; if any black specks are formed, even tem- 
porarily, the mixture is incomplete; a certain amount of green color is, how- 
ever, practically unavoidable. If no black coloration is obtained, the mix- 
ture is satisfactory. Allow the mixture to stand in the mortar over night 
(covered with paper) and place in well stoppered bottles for future use. 

Technic. 

Fill a special sugar buret, graduated in 0.02 c.c, by suction with the 
urine to be tested. These burets should have accessory tips, which are 
drawn out to almost a capillary point in order to permit the delivery of 
a large number of drops to the unit of measure. This precaution does 

1 Jour. Biol. Chem., 1919, XXXVIII, 287. 

2 Ibid., 1918, XXXIII, 513. 

3 Ibid., 1911, IX, 57; Jour. A. M. A., 1911, LVII, 1193. 
* Jour. Biol. Chem., 1919, XXXVII, 303. 



THE URINE 313 

away with the necessity of dilution of highly saccharine urines before making 
the determination. Albumin does not interfere with this test, although it 
does alter the appearance of the precipitate of cuprous thiocyanate which 
forms, rendering it more flocculent and bulky. 

Transfer 5 c.c. of the above copper sulphate solution to a large hard 
glass test-tube. Add approximately i c.c. of the saturated sodium car- 
bonate solution; shake for a moment, and then add 4 to 5 grams of the 
above salt mixture. Heat gently with shaking until all the salts have been 
dissolved, except for a few isolated particles of sodium carbonate. A clear 
solution is usually obtained in less than i minute at temperatures which 
need not exceed 6o°C. Now add the urine, 0.4 to i c.c:, heat fairly rapidly 
to boiling, and then boil very gently so as not to drive off too much 
water. When the mixture begins to bump add a small glass bead. If the 
boiling contents of the test-tube do not suddenly become filled with the 
white precipitate of cuprous thiocyanate within the first 15 seconds of 
boiling, then less than 3^ of the required sugar has been added and more 
of the urine must be added without delay. If the full required amount of 
sugar is present in the urine first added, the solution becomes turbid 5 sec- 
onds after the boiling point has been reached. The only time restriction 
called for in connection with the final titration is that complete reduction, as 
evidenced by decoloration of the solution and precipitation of the white cup- 
rous thiocyanate, must not occur in less than 4 minutes boiling. The 
volume of the solution in the test-tube should not become less than 6 to 7 c.c. 

Five c.c. of this copper solution are reduced by 25 mg. of dextrose or 
levulose, 45 mg. of anhydrous maltose, or 40.4 mg. of anhydrous lactose. 

Bang's Method. 

This method^ appears to the writer to be one of the best at our disposal for 
the quantitative estimation of sugar in the urine. Like the solution of Bene- 
dict previously mentioned, it contains alkali carbonates instead of hydrates. 
The principle of this method is as follows: The urine is treated with an excess 
of|standard alkaline copper solution and boiled to bring out the reduction due 
to sugar. The amount of copper remaining in excess is then determined by 
titration with a solution of hydroxylamin sulphate (instead of with potassium 
sulphocyanate as used by Citron) and from this the amount of sugar is 
calculated. 

The solutions necessary in this test are as follows : 

(a) One hundred grams of potassium bicarbonate are dissolved in about 
1300 c.c. of distilled water contained in a 2-liter flask. To this solution are 
added 500 grams of potassium carbonate and 400 grams of potassium sulpho- 
cyanate. Exactly 25 grams of pure copper sulphate (CUSO4 5H2O) are then 
dissolved in about 150 c.c. of warm distilled water. After cooling this solu- 
tion is added quantitatively to the carbonate solution. Add water up to the 

^ Biochem. Ztschr., 1907, II, 271; 1908, XI, 538; 191 1, XXXII, 443. Bang (Ibid., 1913, 
XLIX, i) has devised a new method which is cheaper and equally exact. See, also, Citron, 
Miinch. Med. Wchnschr., 1918, LXV, 1053. 



314 



DIAGNOSTIC METHODS 



2-liter mark, allow to stand for 24 hours and filter. This solution is stable for 
about three months.^ 

(b). Two hundred grams of potassium sulphocyanate are dissolved m 
about 1500 c.c. of distilled water in a 2-liter flask. 6.55 grams of hydroxy- 



BANG'S TABLE OF REDUCTION EQUIVALENTS 



Cubic centimeters of 


Milligrams of 


Cubic centimeters of 


Milligrams of 


hydroxylamin solution 


sugar 


hydroxylamin solution 


sugar 


0.75 


60.0 


25.50 


23.5 


1. 00 


59.4 


26.00 


22.9 


1.50 


58.4 


26.50 


22.3 


2.00 


57.3 


27.00 


21.8 


2.50 


56.2 


27.50 


21.2 


3 00 


55.0 


28.00 


20.7 


3.50 


54.3 


28.50 


20.1 


4.00 


53.4 


29.00 


19.6 


4.50 


52.6 


29.50 


10. 1 


5.00 


51.6 


30.00 


18.6 


5.50 


50.7 


30.50 


18.0 


6.00 


49.8 


31.00 


17. S 


6.50 


48.9 


31.50 


17.0 


7.00 


48.0 


32.00 


16. 5 


7.50 


47.2 


3!2.5o 


15.9 


8.00 


46.3 


3'3 00 


15.4 


8.50 


45.5 


33.50 


14.9 


9.00 


44.7 


34.00 


14.4 


9.50 


44-0 


34.50 


13.9 


10.00 


43.3 


35.00 


13.4 


10.50; 


42.5 


35.50 


12.9 


11.00 


41.8 


36.00 


12.4 


11.50 


41. 1 


36.50 


II. 9 


12.00 


40.4 


37.00 


II. 4 


12.50 


39.7 


37.50 


10.9 


13.00 


39.0 


38.00 


10.4 


13.50 


38.3 


38.50 


9.9 


14.00 


37.7 


39.00 


9.4 


14.50 


37.1 


39.50 


9.0 


15.00 


36.4 


40.00 


8.5 


15.50 


35.8 


40.50 


8.1 


16.00 


35.1 


41.00 


7.6 


16.50 


34.5 


41.50 


7.2 


17.00 


33.9 


42.00 


6.7 


17.50 


33-3 


42.50 


6.3 


18.00 


32.6 


43.00 


5.8 


18.50 


32.0 


43.50 


5.4 


19.00 


3' -4, 


44.00 


4.9 


19.50 


30. 8J 


44.50 


4.5 


20.00 


30.2 


45.00 


4.1 


20.50 


29.6 


45.50 


3.7 


21.00 


29.0 


46.00 


3-S 


21.50 


28.3 


46.50 


2.9 


22.00 


27.7 


47.00 


2.5 


22.50 


27.1 


47.50 


2.1 


23.00 


26.5 


48.00 


1.7 


23-50 


25.8 


48.50 


1-3 


24.00 


25-2 


49.00 


0.9 


24.50 


24.6 


49.50 


0.5 


25.00 


24.1 


50.00 


0.0 



For every }/[ q c.c. hydroxylamin solution used more than is given in the table, subtract 
0.1 mg. if the reading be between 49 and 15, while if it be between 15 and i subtract 0.2 mg. 

^ Hatta (Biochem. Ztschr., 1913, LII, i) shows that the self-reduction of this solution 
may give as high an error as 9% on the positive side. 



THE URINE 315 

lamin sulphate are dissolved in water and added quantitatively to the sulpho- 
cyanate solution. Add water up to the 2-liter mark and preserve the mix- 
ture in dark colored bottles. This solution is very permanent. 
Technic. 

Ten c.c. of urine (5 or 2 c.c. diluted with water to 10 c.c. if more than 0.6 
per cent, sugar is present) are measured into a 200 c.c. Jena Erleiimeyer flask 
and treated with 50 c.c. of the copper solution (a). Heat on a wire gauze to 
boiling for exactly three minutes. Cool quickly to room temperature by im- 
mersion of the flask in cold water. Now titrate with the hydroxylamin solu- 
tion (b) until a colorless mixture results. From the number of c.c. of hydroxy- 
lamin sulphate solution used, calculate the sugar in milligrams by means of 
the accompanying table. A simple calculation yields the percentage and 
total amounts. 

It has been found difficult to get a sharp end-point in this reaction if the 
urine be rather highly colored. Bang and Bohmansson^ have shown that the 
urine may be cleared by the following process. Twenty c.c. of urine are 
treated with 5 c.c. of 25 per cent. HCl and 2 grams of blood charcoal. Shake 
a few times during five minutes and filter through a dry filter into a dry beaker. 
This filtrate is then used for the test as described above. Woodyatt and 
Helmholz^ and Andersen^ have called attention to the necessity of testing the 
charcoal by control experiments before using the above method of clearing. 
As some of the blood charcoals give very erroneous results, they recommend 
the use of Merck's preparation. A further advantage of using this clearing 
method is that the "auto-reduction" of the urine is reduced to a low limit 
owing to the almost complete removal of urochrom, uric acid, creatinin and 
glycuronic acid derivatives.^ 
Purdy's Method. 

It has been found that the addition of strong ammonia to the mixed copper 
and tartrate solutions makes the end-point much more distinct. Pavy, Sahli, 
Kumagawa and Suto, Kinoshita, and others have modified the original 
Fehling's solution in this way. It was shown, however, by Lowe that the 
substitution of glycerin for the Rochelle salt made a much more stable solution 
and one which could be originally mixed and kept for indefinite lengths of time. 
Purdy has succeeded in obtaining a copper solution which, in the writer's 
experience, is much to be preferred for general laboratory purposes over most 
of the other modifications of Fehling's solution. His formula is a follows: 

Chemically pure copper sulphate, 4-752 grams 

Potassium hydrate,^ 23.500 grams 

Strong C. P. ammonia (sp. g. 0.88), 350.000 c.c. 

Glycerin, 38.000 c.c. 

Distilled water, q. s. ad., 1,000.000 c.c. 

^ Ztschr. f. physiol. Chem., 1909, LXIII, 443; Biochem. Ztschr., 1909, XIX, 281. 
- Arch. Int. Med , 191 1, VII, 598. 
3 Biochem. Ztschr., 191 1, XXXVII, 262. 

^ Bang (Biochem. Ztschr., 1912, XXXVIII, 168) has recently advised the substitution 
of 2 c.c. of 95% alcohol for the 5 c.c. of HCl. 
^ Or 16.8 grams of sodium hydrate. 



3l6 DIAGNOSTIC METHODS 

This solution is prepared by dissolving the copper sulphate and glycerin 
in 200 c.c. of distilled water, heating gently if necessary. The potassium 
hydrate is dissolved in a second 200 c.c. of water and mixed with the copper 
solution. When the mixture has cooled add the ammonia and bring the total 
volume up to i liter with distilled water. Thirty-five c.c. of this solution are 
decolorized by 0.02 gram of glucose. 

Haines has slightly modified this original solution of Purdy so that 10 c.c. 
of the solution are decolorized by o.oi gram of glucose. This titer is some- 
what more convenient than that of Purdy as the calculation is distinctly 
simplified. The formula for his modification is as follows: 

Pure copper sulphate, 8.314 grams 

Pure potassium hydrate,^ 25.000 grams 

Glycerin, ^0.000 c.c. 

' Ammonia, 350.000 c.c. 

Distilled water, q. s. ad., 1,000.000 c.c. 

The principle of this test depends upon the fact that, in the reduction of 
cupric oxid in solutions of definite strength by glucose, the blue coloration 
disappears on the addition of a definite amount of glucose without any attend- 
ant precipitate, the reduced solution remaining transparent and colorless. 
Technic. 

Thirty-five c.c. of Purdy's test solution or 10 c.c. of Haines' solution 
are measured into an Erlenmeyer flask and 50 c.c. of water added. The 
flask is then closed with a doubly perforated rubber stopper, through one hole 
of which passes the stem of a buret containing the suspected urine and 
through the other a bent glass tube to conduct the fumes of ammonia away 
from the observer. The object of closing the flask with the stopper is to 
exclude the air and thus prevent reoxidation of the cuprous oxid. The con- 
tents of the flask are now brought to a gentle boil and the urine added, i c.c. at 
a time, shaking the flask after each addition until the fluid is completely de- 
colorized. The number of c.c. used is then noted and a second test made, 
adding at once i c.c. less of urine than the total number of c.c. used in the first 
experiment. The last portions of the urine are added two drops at a time, 
allowing from three to five seconds to elapse between the addition of these 
separate portions. When the urine is completely decolorized the number of 
c.c. used is noted. As the amount of urine used is equivalent to 0.02 gram of 
glucose with Purdy's solution or o. 01 gram withHaines' solution, the percent- 
age may be obtained as in the previous method. It is necessary with this method 
that the urine be diluted for the same reasons as previously mentioned. 

In all of the copper tests for glucose the influence of preservative agents 
must be remembered. Thus chloroform, chloral hydrate, and formahn will 
all reduce copper solutions so that an error may be introduced unless these 
substances are removed. 

In selecting a method for the quantitative determination of sugar in the 
urine, the general worker should remember that the method used should be both 

^ Or 18 grams of sodium hydrate. 



THE URINE 



317 



simple and exact. With these points in view, the writer has Httle hesitancy 
in recommending FoHn and Peck's or Bang's method. If the urine be cleared 
as previously described, the results are all that could be desired, the error 
being extremely small. Many other modifications of the copper tests have 
been advocated, but the writer does not feel that they have any advantage 
from either the scientific or clinical standpoint. The method of Allihn, in 
which a copper solution is reduced and the precipitated cuprous oxid either 
weighed as such or further reduced to metallic copper in a stream of hydro- 
gen, is not clinically available. The modifications of Rudisch, Rudisch and 
Celler, and of Gerrard and Allan do not give as accurate results as do the 
above methods in the experience of the writer.^ 
Polariscopic Method. 
In this test the urine must be absolutely clear and must contain no albu- 
min. Moreover, it is essential that such substances as glycuronic acid and 
j8-oxybutyric acid be removed before polarization as they will introduce con- 




FiG. 84. — One form of Laurent Polariscope. {Hawk.) 

B, Microscope for reading the scale; C, a vernier; E, position of the analyzing Nicol 

prism; H, polarizing Nicol prism in the tube below this point. 



siderable error in the reading. The best clearing agent is that, previously 
mentioned under Bang's test, of shaking the urine with HCl and blood char- 
coal and filtering. Instead of this method the urine, acidified with acetic acid, 
may be treated with a solution of normal lead acetate, which will precipitate 
the albumin and remove excess of pigment. The mixture is filtered and the 
clear filtrate is used for the test. It must be remembered that a correction 
must be made in the percentage of sugar in the filtrate, if the urine be cleared 
with a solution of lead acetate. This may be done by adding 25 c.c. of a 10 
per cent, solution of lead acetate to 75 c.c. of urine. The sugar in the filtrate 
will represent only three-fourths of that of the original urine. Basic lead acetate 
should be used with caution, as it precipitates various sugars. If the urine con- 
tain no albumin, magnesium oxid or silicic acid may be used as clearing agents. 
Various types of polarimeters have been suggested and most of them 

^ See Nagasaki, Ztschr. f. physiol. chem., 1916, XCV, 61; Wilson and Atkins, Biochem. 
Jour., 1916, X, 504; Vansteenberghe and Bauzil, Paris Med., 1916, VI, 556; 1917, VII, 164; 
Cammidge, Lancet, 1917, I, 613. 



3l8 DIAGNOSTIC METHODS 

are quite satisfactory for the determination of sugar. One of these with 
its description is seen in the accompanying cut and legend. The principle 
upon which their use depends is the fact that optically active substances 
when in solution have the power of turning the plane of polarized light either 
to the right or the left. The zero point of the instrument is determined by 
observing the point at which the halves of the optical field have exactly the 
same degree of illumination, when the Hght passes through a tube either 
erripty or containing an optically negative fluid. The point at which the 
graduated scale of the instrument and the vernier correspond is regarded as 
the zero-point. In the use of the polariscope any deviation of the plane of 
light passing through the polarizing Nicol prism will be noticed by a darken- 
ing of one portion of the field, so that the compensating or analyzing Nicol 
must be rotated until both parts of the field are equally illuminated. In 
this way one readily determines how much the plane of polarized hght has 
been deviated, by observing the degree of rotation necessary to bring the two 
portions of the field into equal illumination when the Hght passes through an 



3 



[oni 



Fig. 85. — Diagrammatic representation of the course of light through the Laurent 

polariscope. (Direction reversed from that of previous figure.) {Hawk.) 

a, Bichromate plate to purify the light; h, the polarizing Nicol prism; c, a thin quartz 

plate covering one-half the field and essential in producing a second polarized plane; d, tube 

to contain the liquid under examination; e, the analyzing Nicol prism;/ and g, ocular lenses, 

optically active fluid. The most rehable instruments for general work are 
known as the "half-shadow" types, but they rarely find a place in the equip- 
ment of the general worker. The examination is usually made in a dark room, 
the light passing through the tube of the polariscope from a sodium flame. 
It must be remembered that the urine may contain substances other than 
glucose which rotate the plane of polarized light. The normal urine is slightly 
levorotatory, the degree varying between 0.05 and 0.18. Albumin is also 
levorotatory and will interfere markedly with the degree of rotation refer- 
able to glucose, unless it be very sHght in amount or be entirely removed. 
Levulose, /?-oxybutyric acid, and the conjugated glycuronates are also levo- 
rotatory, the second being especially prone to interfere with the glucose rota- 
tion as it so frequently is associated with glucose in diabetic conditions. If 
the urine has been previously heated with acid before being examined in the 
polariscope, glycuronic acid will also interfere, as it shows distinct dextrorota- 
tion. Cane-sugar as well as lactose are occasionally found in the urine and 
may lead to confusing results as both are dextrorotatory substances. It is, 
therefore, necessary before any reliable results may be obtained with the 
polariscope to remove the interfering substances, either as preliminary to the 
determination of glucose or subsequently, a correction being then made for 
the rotation of the interfering substance. It is to be said that the polariscope 
is more fitted for a clinical laboratory than for the general practitioner. 



THE URINE 



319 



The rotation of light when passing through optically active solutions is 
dependent upon several factors, among which we find the temperature at 
which the observation is made, the length of tube through which the light 
passes and the concentration of the active solution. By specific rotation of 
a fluid is meant the rotation observed when light passes through a solution 
containing i gram of the active substance per c.c. of fluid and placed in a 
tube one dcm. in length. Thus we find the specific rotation of glucose in 
such a tube being (a)o = +52.74. It is, therefore, evident that the per- 
centage of sugar in an unknown solution contained in a tube i dcm. in length 
may be obtained by dividing the degree of rotation by 0.5274. Some of the 
tubes for clinical purpose are constructed of such a length (188.6 mm.) that one 
degree of rotation equals i per cent, of glucose. For a full discussion of the sub- 
ject of optical activity of fluids the writer would refer to the workofLandolt.^ 
Technic. 

Having determined the zero-point of the instrument, the tube, which 
should be thoroughly cleaned and dry, is filled with the fluid to be examined. 
In filling this tube precautions must be taken not to include any bubbles of 
air, which is best done by filling the tube until a convex 
meniscus is observed and then sliding the glass disk 
over the end in such a way that the excess of fluid is 
shoved off. The metal cap is then screwed on tightly, 
but in such a way that undue compression is not 
exerted upon the glass disk. The tube is placed in 
position, the field distinctly focussed and the degree 
of rotation determined by revolving the analyzing 
Nicol until the two portions of the optical field have 
exactly the same intensity of illumination. This will 
require considerable experience, as the accuracy of 
the determination will depend not only upon the 
clearness of the fluid, the degree of focussing, the 
sensitiveness of the instrument, and the brightness 
of the light, but also upon the sensitiveness of the 
observer's eyes. Several readings should be made, 
turning the prisms from both directions and observing 
the degree at which the fields are isochromatic, the 
average being taken as the final result. As the eyes 
soon become fatigued, they should be used for only a 
few seconds at a time. 

Having determined the degree of rotation, the percentage of sugar is then 
calculated as previously mentioned. This method, if carefully used and 
interfering substances avoided, is the most accurate method for the determina- 
tion of glucose. It is, however, difficult, requires much experience, and is 
not as sensitive as the other methods, rarely detecting the presence of less 
than 0.2 per cent, of glucose. Theoretically, the percentage of sugar as de- 
termined by the polariscopfe should exactly agree with that obtained by titra- 
1 Das optische Drehungs-Vermogen, Braunschweig, 1898. 




Fig. 86. — Einhorn sac- 
charometer. 



320 DIAGNOSTIC METHODS 

tion of copper solutions, but in general work such is never the case, as inter- 
fering substances may be present or have not been completely removed. 

The writer must refer elsewhere for rnethods of correcting the readings 
of the polariscope when interfering substances are present, as he does not 
think it wise to recommend the general worker to waste his time with this 
instrument when sufficiently accurate clinical results may be obtained by 
methods which are for him much easier and less liable to error. 
Fermentation Methods. 

The principle of this method has been previously outlined. It has been 
found that, if the precautions mentioned are observed, carbon dioxid is 
evolved quantitatively from glucose by the action of yeast. The most con- 
venient method of applying the test is to use an Einhorn fermentation tube 
which is so graduated that the amount of CO2 evolved is directly read off in 
terms of per cent, of glucose. For this purpose the urine must contain less 
than I per cent, of glucose, the urine being diluted as previously described 
to bring the amount within this figure, multiplying the result, of course, by 
the degree of dilution. The urine, which should be acid in reaction, is shaken 
with a piece of compressed yeast about the size of a small pea, all precautions 
being observed as mentioned above. The mixture is then poured into the 
fermentation tube in such a way that no air-bubbles collect at the upper end 
of the tube. Controls as previously described are then made and the three 
tubes placed in the incubator at 37°C. In a few hours bubbles of gas (CO2) 
will collect at the top of the tube, the fermentation being practically complete 
overnight. The percentage of sugar is then read direct from the calibration 
(1/4-1 per cent.) on the tube (the figures from one to five representing c.c. 
of gas and not per cent, of sugar). 

Lohnstein's Saccharometer. 

This apparatus is seen in the accompanying cut. Twelve c.c. of mercury 
are placed in the bulb of the apparatus. One-half c.c. of the urine to be 
tested is then floated upon the mercury and treated with a thick paste of com- 
pressed yeast diluted two or three times with water. The stopper is then 
carefully greased with vaseline and inserted so that the two apertures corre- 
spond. By tipping the apparatus a trifle the column of mercury in the long 
tube is then adjusted to the zero point of the scale. When this is done the 
stopper is turned so that the holes no longer correspond and the weight is 
placed on the stopper to prevent leakage from the increased pressure of the 
gas liberated in the fermentation. The apparatus is then placed in the incu- 
bator and the extent of fermentation read off by noticing the height to which 
the column of mercury rises in the long arm of the instrument. The fermen- 
tation is usually complete within six hours. After removing the apparatus 
from the incubator it is allowed to stand in the air for a few minutes to adjust 
itself to room temperature, as the scale is graduated in this way. 

This method gives results which correspond very closely to those of titra- 
tion and is to be recommended for the quantitative determination by 
fermentation methods. 

Lohnstein has also introduced a saccharometer which may be used with 



THE URINE 



321 



dilute urine. It is seen in the accompanying cut. It does not, in the writer's 
opinion, have any advantage over the above-mentioned apparatus as the 
principle is the same, although the urine must be diluted. 
Robert's Method. 
This method has been recommended for the quantitative determination 
of sugar and is based upon the fact that the specific gravity of the urine is 
changed in a quantitative way when the sugar of the urine is fermented. 



ia 



35''c 



Fig. 87. — ^Lohn- 
stein's fermenta- 
tion tube for undi- 
luted urine. 




Fig. 88. — Lohnstein's fermentation 
tube for diluted urine. 



The urine must be acid before applying this test. A piece of yeast about 
the size of a bean is added to the urine which is allowed to ferment at incu- 
bator temperature until no further quahtative test for sugar is obtained. 
This will usually require from 24 to 48 hours, so that a trace of sodium fluorid 
should be added to the urine to prevent bacterial action. The specific 
gravity of the urine before it is subjected to fermentation is very carefully 
taken either with a very accurately standardized hydrometer or, preferably, 
with the pycnometer. After fermentation is complete the specific gravity 
of the fermented urine is determined in the same way. The difference in 
the specific gravity of the two specimens is then multiplied by 234 to obtain 
the percentage of sugar. Or, according to Purdy, each, degree of specific 
gravity lost in fermentation corresponds to i grain of sugar per fluidounce. 

If the specific gravity be accurately determined the results are correct 



32 2 DIAGNOSTIC METHODS 

within O.I per cent. As this method requires, however, the use of a pycnom- 
eter and a very accurate chemical balance, it can hardly be recommended 
to the general practitioner for his work. 

(b) Levulose (d-fructose), CH2OH— (CH0H)3— CO— CH2OH. 

Levulose is found very widely distributed throughout the vegetable king- 
dom, especially in fruits. Honey is almost a pure levulose. It may be found 
in the urine, transudates, or exudates, after a large intake of levulose-con- 
taining food or may occur spontaneously, when the subject has taken little 
such food. The levulosuria, reported by Zimmer, Ventzke, Czapek, Worm- 
Miiller, Seegen, Mauthner, Cotton, Roehmann, Personne, Henninger, Marie, 
and Robinson, must be accepted with reserve, as the incompleteness of the 
methods then in use afforded no certain means of recognizing levulose (Neu- 
berg^). The most authentic cases of true levulosuria are those of May, 
Schlesinger, Rosin and Laband, Lepine and Boulud, and Neubauer. This 
pure levulosuria occurs in both sexes and at all ages, the amount of sugar 
excreted being subject to variations from 2.7 grams per diem (Schlesinger) 
to 24 grams (Lepine). 

More common than pure levulosuria is its association with a glycosuria. 
This combination appears in all forms of diabetes, in the severe types leyulose 
being practically never missed, according to Umber, especially when no 
restriction is placed on the carbohydrate intake. Neubauer's observations 
on this point are interesting. He finds that withdrawal of carbohydrates 
causes both levulose and dextrose to disappear from the urine. When 
levulose was given it was utilized, but when glucose was taken it was less 
completely assimilated, being excreted in part as levulose. 

The tolerance for levulose is, as a rule, less than that for glucose, so that 
we are not surprised to find that the administration of 100 grams of levulose 
to normal individuals is followed by a levulosuria in about 10 per cent, of 
cases, while no such effect may be observed in diabetes mellitus. Here the 
question of individual tolerance must be considered, as Umber finds 25 grams 
of levulose excreted following an intake of 100 grams, while the writer has 
observed an excretion of 75 grams on the same intake. Strauss^ finds that 
an alimentary levulosuria occurs, after an intake of 100 grams of levulose, 
in 90 per cent, of cases of functional hepatic disturbance. This test would 
seem, therefore, to be a valuable indication of hepatic insufficiency, although 
it is not pathognomonic. Also alimentary galactosuria, following the adminis- 
tration of 40 grams of galactose to the fasting stomach, as originated by 
Bauer, 2 has been shown by Reiss and Jehn and, also by Roubitschek to be 
inconstant. . A decidedly positive excretion of galactose is observed only in 
cases in which the entire liver parenchyma is presumably involved, as in the 
acute infections and intoxications characterized by catarrhal jaundice. 

^ Handbuch der Pathologic des Stoffwechsels, Berlin, 1907, 716; also, Cammidge and 
Howard, Lancet, 1915, I, 320. 

2Deutsch. med. Wchnschr., 1901, XXVII, 757 and 786; Ibid., 1913, XXXIX, 1780. 
See, also Oszacki and Wagner, Med. Klin., 1913, IX, 1549; Draudt, Arch. f. exper. Path. u. 
PharmakoL, 1913, LXXII, 457; Hohlweg, Miinch. med. Wchnschr., 1913, LX, 2271; and 
Schirokauer, Ztschr. f. klin. Med., 1913, LXXVIII, 462. 

3 Deutsch. med. Wchnschr., 1908, XXXIV, 1505. 



THE URINE 323 

Cases of cirrhosis, carcinoma, cholelithiasis and phosphorus poisoning react 
either negatively or very slightly.^ 

The chemical reactions of le-\^ulose are very similar to those of dextrose. 
Owing to the presence of the ketone group in its molecule, it shows the same 
reducing actions as does the aldehyd group of the glucose. Like glucose it 
ferments, but not quite so readily. It is levorotatory, its specific rotation 
being (a) ^ = —91 degrees. Leo has reported the finding of a levogyrate carbo- 
hydrate in diabetic urines, which he believes to be laiose. This differs from lev- 
ulose in being unfermentable with yeast. Levulose forms exactly the same 
phenylosazon as does glucose, so that it is a matter of great difficulty to differ- 
entiate these bodies by the tests given above. Lobry de Bruin and Alberda 
von Eckenstein have shown that glucose and levulose may change, the one 
into the other, by the action of traces of alkali, acids, and neutral salts, such 
as sodium acetate. 

Seliwanoff's Test. 

This test has been advanced as one characteristic for the ketoses in dis- 
tinction from the aldoses. Ten c.c. of urine are treated with a few crystals 
of resorcin and 5 c.c. of concentrated HCl. If the mixture be warmed a bril- 
liant red color appears in the presence of a ketone (levulose) while no colora- 
tion is observed with an aldehyd (glucose).^ Miiller has shown that gluco- 
samin gives this test, while R. and O. Adler find a reaction in the presence of 
nitrous acid. If the mixture be heated too strongly or too long, mannose and 
maltose may also give a positive test. Adler finds that the use of acetic acid 
with a trace of HCl gives better results than HCl alone. It will be seen, there- 
fore, that this test is not so characteristic as was beheved, but it serves to 
distinguish levulose from glucose, which is the important point. 

If the red solution formed in this reaction be neutralized with sodium 
carbonate and extracted with amyl alcohol or, preferably, with acetic ether 
(Borchardt), the extract will have a yellow color with a faint green fluores- 
cence and becomes rose-red on the addition of alcohol. The spectrum of this 
solution shows a sharp line in the green between E and h, while if the solution 
be quite concentrated a second weaker line will be seen in the blue at F. 
Methyl-phenylhydrazin Test. 

Neuberg^ has definitely shown that fructose forms a characteristic osazone 

^ Reiss and Jehn, Deutsch. Arch. f. klin. Med., 191 2, CVIII, 187; Roubitschek, Ibid., 
226; Foster, Am. Jour. Med. Sc, 1912, CXLIII, 830; Editorial, Jour. Am. Med. Assn. ,"1913, 
LX, 287; Bloomfield and Hurwitz, Bull. Johns Hopkins Hosp., 1913, XXIV, 375; Hurwitz 
and Bloomfield, Ibid., 380; Isaac, Ztschr. f. physiol. Chem., 1914, LXXXIX, 78; Wagner 
Ztschr. f. klin. Med., 1914, LXXX, 174; Hatiegan, Wien. klin. Wchnschr., 1914, XXVII, 
358; Fleckseder, Ibid., 475; Aral, Deutsch. med. Wchnschr., 1914, XL, 792; Worner and 
Reiss, Ibid., 907; Maliwa, Med. klin., 1914, X, 762; Friedman and Strouse, Arch. Int. Med., 
1914, XIV, 531; Hoffmann, Ztschr. f. exper. Path. u. Therap., 1914, XVI, 337; Schede, 
Jahrb. f. Kinderhkde., 1915, LXXXII, 45; Pari, Gaz. d. Osp., 1916, XXXVI, 1217; Foster 
and Kahn, Jour. Lab. and Clin. Med., 1916, II, 25. 

2 Konigsfeld (Biochem. Ztschr., 1912, XXXVIII, 310) shows that the HCl must not 
have a final concentration greater than 12.5 per cent, and that the heating should not be 
longer than 20 to 30 seconds. If dextrose, also, be present in amount greater than 2 per 
cent., the possibility of its conversion into levulose must be remembered. See, also, Jolles, 
Ibid., 1912, XLI, 331. 

3 Ztschr. f. physiol. Chem., 1902, XXXVI, 227; Ber. d. d. chem. Ges., 1902, XXXV, 
959; Ibid., 1904, XXXVII, 4616. 



324 DIAGNOSTIC METHODS 

with methylphenylhydrazin, while no such compound is obtained with glucose, 
mannose, or glucosamine. The test is, therefore, the most reliable and scien- 
tific one for the presence of levulose, although it is not clinically so acceptable 
as the Seliwanoff reaction. 

The formation of this osazone occurs according to the following equation. 

CH2OH - (CH0H)3 - CO - CH2OH + 2NH2 - N(CH3) (CeHs) = CH2OH 
- (CH0H)3 - C CH + 2H2O + 2H 

II 11 

(CH3) (C6H5)N - N N - N(CH3) (CeHs) 

Technic. 

The urine is acidified with acetic acid and boiled to remove albumin if 
present. The mixture is then filtered and the clear filtrate, which must be 
acid, is evaporated in a vacuum, at a temperature not over 40°, to a thin 
syrup. The reaction must remain acid during the evaporation so that a drop 
of acetic acid may be added if necessary. The residue is thoroughly extracted 
with 98 per cent, alcohol, using an amount of alcohol equal to one-half the 
original volume of urine. Filter and re-extract the residue with alcohol 
should any reducing action be observed in it. The alcoholic extracts are 
mixed and decolorized with animal charcoal. A portion of the extract is ex- 
amined for its sugar-content by Fehling's test, all of the reduction being 
attributed to levulose. Methylphenylhydrazin is then added to the alcoholic 
solution, which should not measure over 30 c.c. The amount of the hydrazin 
to be added is in the proportion of 3 molecules for i of levulose or, in other 
words, for each gram of sugar a trifle over 2 grams of methylphenylhydrazin. 
The mixture is allowed to stand for a few hours in the cold and filtered if a 
precipitate forms. The filtrate is treated with 50 per cent, acetic acid, using 
the same amount of acid as of the methylphenylhydrazin, and sufficient 
alcohol is added to give a clear solution. The mixture is heated from three 
to five minutes or, preferably, allowed to stand at 4o°C., for 24 hours in an 
incubator. Crystals will usually separate out at the end of this time, but 
if not they will appear on the addition of a few drops of water. The crude 
product is purified by recrystallization either from a mixture of chloroform 
and petroleum ether or from hot water to which pyridin has been added. 
The yield by this method is 81 per cent, of the total sugar if pure solutions 
are used, while from urine it is but 50 per cent. 

The crystals of methylphenylosazone are delicate yellow, long, fine, needles 
melting from 158 to i6o°C. This method can hardly find application in 
the hands of the general practitioner. 

Pure levulosuria is recognized by the levorotation of the urine, which 
possesses reducing properties and is capable of fermenting with yeast. After 
fermentation, the urine loses its reducing and optical properties. The 
presence of a levulosuria is indicated by a considerable difference between the 
results obtained by titration and polarization, providing a glycosuria be 
coexistent. The Seliwanoff reaction should be used as a routine in every case 
which shows both fermentation and reduction. The general practitioner 



THE URINE 325 

will rarely have access to a polariscope and even then may be misled if other 
interfering substances, such as albumin, glycuronic acid, and jS-oxybutyric 
acid be present. Do not presuppose that glucose is the only fermentable 
and reducing sugar of the urine, as the recognition of the true condition may 
make much difference in the treatment as well as in the prognosis. 

(c) Pentose. 

The pentose^ group of carbohydrates comprises eight possible stereo- 
isomers with the general molecular formula of CsHioOs. Three members 
of this group, rhamnose, fucose, and chinovose, are substituted pentoses, viz., 
methyl pentoses having the formula C6H12O5. These latter are the excep- 
tions to the general rule that carbohydrates contain the same number of car- 
bon atoms as of o^xygen atoms, and have led to the more scientific method of 
classifying the carbohydrates according to the number of oxygen atoms rather 
than of carbon atoms. The pentoses are widely spread throughout the vege- 
table kingdom in the form of their anhydrids or in combination with other 
groups of atoms. They* are also found as constituents of the nucleoproteins 
of animal tissues, being especially abundant in the pancreas. 

It has been found that the ingestion of large amounts of pentose-contain- 
ing food, such as apples, cherries, plums, beets, and the leguminous vege- 
tables, leads to the excretion of pentose in the urine. This alimentary type of 
pentosuria is characterized by the presence of optically active xylose or arabi- 
nose and appears after the ingestion of small amounts, in some cases follow- 
ing an intake of as low as 50 mg. of the pure carbohydrate. In true diabetes 
mellitus the urine frequently contains 1-xylose which probably arises from the 
breaking down of the pancreatic nucleoprotein. Alfthan states that the 
pentoses are constantly present in diabetic urine, so that it is highly probable 
that these sugars might be found whenever search was made for them in 
diabetic conditions. It is not strange that these carbohydrates are not more 
frequently reported, as their reducing action leads to confusion unless con- 
trolled by fermentation methods. The excretion of pentoses in diabetic 
conditions is not necessarily increased in direct proportion to the intake, as 
their absorption may be so slow that accumulation is not possible before 
oxidation has occurred. 

Salkowski and Jastrowitz^ reported in 1892 the finding of a pentosuria 
which was not associated with intake of pentose food nor with diabetes 
mellitus. This type is known as idiopathic, essential, or intrinsic pentosuria, 
of which 24 cases were found by Janeway^ up to 1906. The peculiar thing 
of this type of pentosuria is that the sugar excreted is r-arabinose, an optically 
inactive pentose. This is the single exception in which an optically inactive 
pentose is found in all nature. This fact characterizes this type of, pen- 
tosuria as an anomaly of metabohsm sui generis. The origin of this urinary 
pentose is still unsettled. The source must be within the organism, as no 
inactive arabinose is taken as food, and if it be given in experimental cases 
it appears in the urine as d-arabinose. Moreover, it cannot be derived from 

^ See Bendix, Die Pentosuria, Stuttgart, 1903. 

2 Centralbl. f. d. med. Wissensch., 1892, 337. 

3 Am. Jour. Med. Sc, 1906, CXXXII, 423. 



326 DIAGNOSTIC METHODS 

the nucleoprotein as the pentose in these cases is 1-xylose.^ Neuberg sug- 
gests that galactose might be considered the source of this pentose, but no 
proof of this has been forthcoming. 

In the true idiopathic pentosuria the assimilation of other carbohydrates 
is unchanged and does not influence in any way the excretion of r-arabinose, 
although the active types of this pentose may be excreted at the same time 
in the urine. It is interesting to find that the pentoses taken in as food are 
excreted in different proportions by the diabetic and nondiabetic subjects. 
Thus von Jaksch observes that diabetics excrete from 49 to S2 per cent, of 
arabinose of the food and nondiabetics i to 47 per cent., while nondiabetics 
excrete from 19 to 55 per cent, of xylose and diabetics only a trace. The 
amount of pentose excreted in essential pentosuria has been reported as vary- 
ing between 0.08 and i per cent. Neuberg has recently shown that a certain 
amount of the r-arabinose is combined with urea in the form of a ureid, which 
does not reduce Fehling's solution until it undergoes hydrolysis with acid. 
For this reason he believes that the amount of pentose reported is in practi- 
cally all cases 100 per cent, too low. Luzzatto^ has reported the excretion 
of the optically active 1-arabinose entirely independent of the food intake. 

' The pentoses reduce copper solutions as do other carbohydrates, but the 
reduction is much slower, appearing during the cooling of the fluid. Five 
c.c. of Folin's solution are reduced by 0.0271 gram of pentose. They do not 
ferment with yeast and do not give a typical reduction with the Almen-Ny- 
lander test, the color being a gray rather than a black. In the true iodio- 
pathic pentosuria no reaction is observed with the polariscope, while in the 
alimentary type a slight dextrorotation is usually noted, although von Jaksch 
reports the excretion of an inactive arabinose after the ingestion of active 
pentoses. These pentoses form more or less typical osazones which melt 
between 157 and 160°, but the reaction is not so easily produced.^ These 
pentosazones are readily soluble in warm water and show dextrorotation. 
The most characteristic chemical property of these types of carbohydrate 
is the formation of furfurol (C2H2O2) when they are distilled in the presence 
of acids. The color reactions given below are based upon the production 
of furfurol and the formation of distinct colorations on treatment with 
various reagents. 

Tollen's Test. 

A few c.c. of concentrated hydrochloric acid are saturated with phloro- 
glucin, care being taken to leave a small amount undissolved. This solution 
is then divided into two equal parts, to one of which is added 1/2 c.c. of the 
suspected urine and to the other 1/2 c.c. of normal urine. Both tubes are then 
placed in a boiling water-bath for a few minutes, w^hen an intense red zone 

^ Elliott and Raper (Jour. Biol. Chem., 1912, XI, 211; Ibid., 1913, XV, 481) suggest the 
possibility of this sugar being ribose. 

2 Beitr. z. chem. Physiol, u. Path., 1905, VI, 87. 

3 Klercker, Deutsch. Arch. f. klin. Med., 1912, CVIII, 277; Levene and La Forge, Jour. 
Biol. Chem., 1913, XV, 481; Ibid., 1914, XVIII, 319; Rosenbloom, Jour. Am. Med. Assn., 
1915, LXIV, 508; Zlataroff, Ztschr. f. physiol. Chem., 1916, XCVII, 28; HiUer, Jour. 
Biol. Chem., 1917, XXX, 129; Fred, Peterson, and Davenport, Ibid., 1919, XXXIX, 347. 



THE URINE 327 

will appear in the upper portion of the tube if pentose be present. After a few 
moments this red color will gradually spread throughout the fluid, while the 
control urine shows no marked change in color. • It is advisable to remove 
the tubes from the water-bath as soon as the color appears, as the clearness 
of the reaction is interfered with by prolonged heating. The coloring matter 
is then extracted by shaking with amyl alcohol, when spectroscopic examina- 
tion will show an absorption band between D and E. 

This test reacts in the same way with glycuronic acid so that it has little 
value in differentiating pentose from the former substance. As a rule, the 
free glycuronic acid is not so easily spHt from its conjugated compound as is 
furfurol from pentose, so that the test is at least suggestive of pentose. 

Orcin Test 

For this test the urine should be decolorized by heating with animal char- 
coal and filtering. Five c.c. of urine are treated with an equal volume of con- 
centrated hydrochloric acid and a few crystals of orcin are added. The mix- 
ture is then warmed approximately to the boiling-point, when a dark green 
color appears in the presence of pentose or glycuronic acid. The formation 
of a greenish-blue precipitate is very strong evidence of pentose rath€r than 
glycuronic acid. The pigment is then extracted with amyl alcohol, when 
spectroscopic examination shows a characteristic absorption band between 
C and D. The presence of glucose may interfere with the reaction, so that 
it may be necessary to remove it by fermentation. 

Bial has modified this test in such a way that glycuronic acid is less apt 
to be a disturbing factor. His reagent consists of 500 c.c. of 30 per cent. HCl 
to which are added i gram of orcin and 25 drops of 10 per cent, ferric chlorid 
solution. Four to five c.c. of this reagent are heated to boiling and removed 
from the flame. The suspected urine is then added drop by drop, not ex- 
ceeding I c.c. in all, when a green color should appear almost immediately if 
pentose be present. The heat employed is hardly sufficient to split off 
glycuronic acid. Here also glucose, if present, should be removed by fermen- 
tation with a pure culture of yeast rather than with compressed yeast, as the 
bacteria possibly present in yeast may break up the pentoses at the same time. 

The osazone may be formed as previously given under Glucose. The 
glucosazone is separated from the pentosazone by digesting with water not 
over 60° in temperature, the pentosazones being dissolved. If the pentosa- 
zone be treated with 20 c.c. of water and 5 c.c. of concentrated hydrochloric 
acid and distilled, the distillate will give a beautiful test with Bial's reagent, 
which absolutely ehminates glycuronic acid and other interfering substances 
possibly present in diabetic urine. 

Quantitative Determination. 

Neuberg and Wohlgemuth^ have introduced a method by which the ara- 
binose of the urine may be accurately determined. A preliminary determina- 
tion of the sugar present is made by Purdy's solution, eliminating glucose 
by previous fermentation. If less than i per cent, of reducing sugar, which is 

1 Ztschr. f. physiol. Chem., 1902, XXXV, 31 and 41. 



328 DIAGNOSTIC METHODS 

assumed to be arabinose, is present, the urine must be concentrated in a 
vacuum so that the sugar content is shghtly over i per cent. 
Technic. 
One hundred ex. of urine are acidified with two drops of 30 per cent, 
acetic acid and evaporated on a water-bath to approximately 40 c.c. It is 
then treated with 40 c.c. of 96 per cent, alcohol, the mixture is allowed to stand 
for two hours, and is then filtered from the separated urates and inorganic 
salts. The residue is carefully washed with 40 c.c. of 50 per cent, alcohol. 
To the filtrate 1.4 grams of pure diphenylhydrazin are added and the mix- 
ture heated on a boiling water-bath for one-half hour, the alcohol being re- 
placed as it evaporates. The mixture is allowed to stand for 24 hours and is 
filtered through a Gooch filter, using the mother liquor to transfer the precipi- 
tate. The crystals are then washed with 30 c.c. of 30 per cent, alcohol, and 
the Gooch with its contents dried at 8o°C. to constant weight. The amount 
of arabinose is obtained by multiplying the weight of the diphenylhydrazone 
by 0.4747 or by dividing by 2.107. 

Cammidge's Reaction. 

Cammidge^ has found that the urine in cases of pancreatic disease contains 
a substance which gives an osazone when treated with phenylhydrazin. In 
his earlier work he was led to believe that this substance was possibly glycerin 
or a derivative. He advanced two reactions, the first of which he found to be 
due to a mixture of glycuronic acid and a true carbohydrate, while the second 
was apparently due only to the glycuronic acid. In his later studies on this 
reaction he was led to believe that the mother substance of the osazone was a 
pentose (probably xylose) split off from a dextrin-like body by the hydrolysis 
with hydrochloric acid. This view has been somewhat substantiated by 
others. The careful work done in recent years by Kinney, ^ Whippel and 
King,^ Whipple, Chaffer and Fischer,* Swan and Gilbridge,^ Wilson,^ and 
Roper and Stillman^ has proven conclusively that, although this reaction is 
given in some cases of pancreatitis, it is not pathognomonic as it occurs in 
many other conditions and may not, even, be present in pancreatic disease.* 
This test is not to be regarded, therefore, as of much diagnostic value. 

Technic. 

The writer gives only the improved method known as "Reaction C 
referring to the original work of Cammidge for reactions A and B. The urine 
to be tested should be a portion of the 24-hour specimen and must be freed 
from glucose and albumin by methods previously outlined. 

^Lancet, 1904, I, 782; Ibid., 1905, II, 14; Robson and Cammidge, Surgery of the Pan- 
creas, London, 1907. See Cammidge and Howard, Lancet, 1914, II, 791; Pekelhering and 
van Hoogenhuyze, Ztschr. f. physiol. Chem., 1914, XCI, 151; Lameris and van Hoogen- 
huyze, Nederl. Tijdschr. v. Geneesk., 1914, II, 675. 

2 Am. Jour. Med. Sc, 1910, CXL, 878. 

2 Bull. Johns Hopkins Hosp., 1910, XXI, 196. 

''Ibid., 1910, XXI, 339. 

•^ New York Med. Jour., 1910, XCI, 781. 

^ Trans. Assoc. Amer. Phys., 1910. 

^ Arch. Int. Med., 191 1, VII, 252. 

* See Mayesima, Mitt. a. d. Grenzgeb. d. Med. u. Chir., 191 2, XXV, 403; also, Karas, 
Ztschr. f. klin. Med., 1913, LXXVII, 125. 



THE URINE 329 

Forty ex. of clear, filtered acid urine are acidified with 2 c.c. of concen- 
trated HCl and boiled for 10 minutes. The mixture is then cooled and made 
up to 40 c.c. with distilled water. The excess of acid is then neutralized 
by the addition of 8 grams of lead carbonate and the mixture cooled if neces- 
sary. Filter off the resulting precipitate and treat the filtrate with 8 grams 
of powdered tribasic lead acetate to remove the glycuronic acid. Filter, 
treat filtrate with 4 grams of powdered sodium sulphate, heat to the boiling- 
point, and allow to cool. The lead sulphate is removed by filtration. Ten 
c.c. of the clear filtrate are made up to 17 c.c. with distilled water, 0.8 gram 
of phenylhydrazin, 2 grams of sodium acetate, and i c.c. of 50 per cent, acetic 
acid are added and the mixture boiled for 10 minutes. Filter while hot and 
make the filtrate up to 15 c.c. with warm water. The mixture is allowed to 
cool, when yellow crystals arranged in sheaves and rosettes may be observed 
under the high-power lens. 

(d) Lactose (C12H22O11). 

Lactose is found in the urine of women during the period of lactation 
and may be found in patients who have been on an exclusive milk diet for a 
long period. A distinct type of alimentary lactosuria is observed on account 
of the low assimilation limit for milk-sugar. In breast-fed children with 
gastrointestinal disturbance lactose associated with galactose may be found 
in the urine. In this case Langstein and Steinitz^ have shown that the excre- 
tion is not due to failure of the normal enzyme, but to an unknown derange- 
ment of the activity of lactase, which renders it incapable of splitting up the 
whole of the lactose, the remainder being absorbed unchanged. From the 
portion which is split up in the bowel the resulting easily assimilable glucose 
is utiHzed by the organism, while galactose partly escapes by the kidneys, 
on account of the much lower assimilation limit for this latter carbohydrate. 
Lactose in these cases is usually associated, therefore, with galactose (Neu- 
berg). According to the work of Voit, an increase of lactose in the diet of a 
diabetic is associated with an increased output of glucose. 

The usual form of lactosuria is that observed in the parturient female. 
It is ordinarily first seen a few days after delivery of the child, but occasion- 
ally appears during the latter days of gestation, as Ney, Lemaire, and Porcher 
have shown. The amount of lactose excreted by the nursing mother equals 
2 to 3 per cent., according to Naunyn, while McCann places the average at 
0.35 per cent, for the first few days of the puerperium. Lactose may continue 
in the urine for some time, the actual amount depending upon the quantity of 
milk as well as its quality.^ If nursing is interrupted for any reason, more 
lactose will be found than when nursing is regular. 

Lactose reduces copper solutions, although somewhat less actively than 
does glucose. It also shows a positive Almen-Nylander reaction. It has'a 
strong rotatory power, its specific rotation being practically the same as that 
of glucose (+ 52.5°). It does not ferment with yeast, although bacteria if 

1 Beitr. z. chem. Physiol, u. Path., 1906, VII, 575. 

2 See Gronvall, Biochem. Ztschr., 1912, XL, 145; Rosenbloom (Jour. Am, Med, Assn., 
191 5, LXIV, 508) reports the finding of lactose in the urine of an adult male after intake of 
milk. 



:^S<^ DIAGNOSTIC METHODS 

present may hydrolyze it into its constituents, glucose and galactose, the 
former of which will show fermentation. It is, therefore, advisable when 
applying the fermentation test not to judge of a reaction which has progressed 
longer than a few hours. With phenylhydrazin it forms a lactosazon which 
appears in the form of sheaves of delicate curved needles much resembhng 
bunches of yellow thread. These crystals melt at 2oo°C. The test is not 
easily obtained unless the urine be concentrated to a small bulk and the 
residue extracted with alcohol, when the alcohol is evaporated and this 
residue taken up with water and the phenylhydrazin test then applied. 
Rubner's Test. 

Ten c.c. of urine are treated with an excess (3 grams) of lead acetate and 
boiled for a few minutes.^ The yellomsh or brown solution is then filtered 
and ammonia added to the filtrate until a slight permanent precipitate 
remains. An intense brick-red fluid is obtained which later shows the depo- 
sition of a cherry-red precipitate with a colorless supernatant fluid. This test 
is not very delicate as it shows lactose only when present in amounts varying 
from 0.3 to 0.5 per cent. Glucose gives with this test a red solution, but a 
more distinctly yellow precipitate. 

Lactosuria is to be assumed when the urine possesses reducing properties 
and dextrorotation, but is incapable of fermenting with ordinary yeast within 
12 hours. If the urine be boiled with 2 per cent, sulphuric acid and then neu- 
tralized, its optical activity will be increased and it will be capable of under- 
going fermentation. It is to be remembered in testing for the amount of lac- 
tose by the use of Folin's or Purdy's solutions that 5 c.c. of the former and 
35 c.c. of the latter are reduced by 0.0339 ^^'^ 0.02712 gram respectively. 

(e) Maltose (C12H22O11). 

Maltose has occasionally been reported in the urine, although many of the 
cases are questionable as the proper identification of the sugar was not 
thoroughly carried out. The most rehable cases appear to be those of Noble, 
von Ackeren, Rosenheim and Flatow, and especially that of Magnus-Levy. 
In this latter case the urine showed a considerable excess of rotation when 
compared with its reducing power. After inversion with dilute acid, by 
which each molecule of maltose was converted into two molecules of glucose, 
the rotation diminished and the reduction increased, so that the polarimetric 
and titration methods gave concordant results. The urine underwent com- 
plete fermentation with synchronous loss of optical activity and of reducing 
power. Calculations founded on these determinations showed that 1.5 per 
cent, of maltose and 2 per cent, of glucose were present. This seems to be a 
case in which the amount of maltose excreted exceeds all records (Neuberg). 
The cases in which maltose appears in the urine seem to be those of disease of 
the pancreas, especially those with interstitial lesions. 

Maltose reduces copper solutions, but not as strongly as does glucose. 
Five c.c. of Folin's and 35 c.c. of Purdy's solution are completely reduced by 
0.04035 and 0.03228 gram respectively of maltose. It is much more strongly 
dextrorotatory than glucose and forms an osazone which crystallizes in 

^See Cole, Biochem. Jour., 1914, VIII, 134; also, Rogerson, Ibid., 1915, IX, 245. 



THE URINE 331 

large prism-like needles arranged in sheaves, and melts at 207°C. This 
osazone is soluble in water and shows a dextrorotation, it being more dis- 
tinctly identified by determination of the nitrogen content, which should 
equal 10.6 per cent. Maltose ferments with yeast only after inversion 
by heating with acid, the splitting products being two molecules of glucose. 

Other carbohydrates, such as dextrin, isomaltose, and saccharose, have 
been reported in the urine. These are extremely rare and need little comment 
in this place. In the case of cane-sugar the assimilation limit is so high that 
an alimentary saccharosuria could occur only after an enormous intake. 
Spontaneous excretion of cane-sugar has never been actually proven, but 
this sugar may be found in the urine of hysterical patients who have added 
it to deceive the physician. The so-called animal gum, first isolated by Land- 
wehr, seems to be a normal constituent of urine. Alfthan^ finds it is present 
in practically every case of diabetes to the extent of i to 37 grams per diem. 
This substance is probably not a definite chemical body, but a mixture of 
several. Little is known of its chemistry. 

Inosite was regarded for a long time as a carbohydrate, but it is now 
known to be a hexaoxyhexahydrobenzol with the formula C6H6(OH)6. This 
substance, has, therefore, nothing to do with true carbohydrate metabolism, 
but is discussed at this point as it has so long been regarded in this connection. 
Inosite enters into the composition of almost all animal tissues and occurs 
both in the optically active and inactive forms. A physiologic excretion of 
inosite is not infrequent, according to Hoppe-Seyler. It may occur in neph- 
ritis, diabetes mellitus and insipidus, and after a large intake of animal food.^ 

(/) Glycuronic Acid (CHO— (CH0H)4-C00H). 

Glycuronic acid is an intermediate product of the oxidation of carbo- 
hydrate, the CH2OH group being converted into CHO while the original 
CHO group is oxidized into COOH. This acid still retains the aldehyd 
group, in consequence of which it shows the same reducing action as does 
glucose. It seems to be characteristic of glycuronic acid that, when pro- 
duced naturally, it is never found in the free state, but only in the combined 
form as the conjugated glycuronic acid. It seems to be especially capable 
of combining with substances showing alcoholic or phenolic characteristics.^ 
The free glycuronic acid may be split off from its conjugated compounds 
by heating with acid and other hydrolyzing agents. The conjugated glycuro- 
nates are levorotatory while the free acid shows dextrorotation. Among 
the substances with which glycuronic acid combines we find chloral hydrate, 
butyl chloral hydrate, chloralamid, camphor, menthol, carbolic acid, resorcin , 
acetanilid, antipyrin, phenacetin, pyramidon, sandal-oil, morphin and 
cocain. The normal metabolism following intake of any of these substances 
is such that excretion of conjugated glycuronic acids will follow and may, 
therefore, lead to the assumption of sugar in the urine unless precautions are 

1 Ueber dextrinartige Substanzen in diabetischen Harn, Helsingfors, 1904. Cammidge 
and Howard, Lancet, 1914, II, 791. 

^ See Anderson and Bosworth, Jour. Biol. Chem., 1916, XXV, 399; Greenwald and Weiss, 
Ibid., 191 7, XXXI, I. See OKey (Jour. Biol. Chem., 1919, XXXVIII, 7,3 and XXXIV, 
149) for a study of the behavior of inulin in the animal body. 

^ See Hamalainen, Skand. Arch. f. Physiol., 1913, XXX, 196. 



332 DIAGNOSTIC METHODS 

taken properly to differentiate these compounds. Besides conjugated gly- 
curonic acid of the above type we find a combination of urea with glycuronic 
acid as well as a certain amount of indoxyl, skatoxyl, phenol, and cresol in 
combination with this acid. Most of the products of bacterial decomposition 
in the intestine are excreted in combination with sulphuric acid, but some is 
invariably present as a conjugated glycuronate. 

The origin and formation of glycuronic acid within the system is not 
entirely understood. It has been supposed to be derived from protein as 
especially advocated by Loewi, but Mayer has rather disprovenLoewi's work, 
and shows that probably glycuronic acid is a direct derivative of glucose and 
that all carbohydrate oxidation must pass through the intermediate stage of 
glycuronic acid. 

The status of this question is very well summed up by Neuberg as follows : 
The formation of glycuronic acid out of protein is by no means excluded, nor 
yet from fat; but as it is difficult to eliminate the direct formation of gly- 
curonic acid or its secondary development from previously existing grape 
sugar, Mayer justly contends that the question of glycuronic acid forma- 
tion from these substances is practically included in the broader question 
of the formation of sugar from fat and protein. 

The exact point of conjugation of glycuronic acid is unsettled. It 
has been assumed by some to occur in the liver, while others find that the 
liver plays no part. It is probable that the synthesis takes place in various 
parts of the organism. 

It has been shown that the output of glycuronic acid may be increased in 
diabetes mellitus, in mild cases the unoxidized sugar being present largely 
in this form. Mayer^ advances the hypothesis of incomplete oxidation of 
sugar to explain its appearance in these cases. ^ He shows that after the ad- 
ministration of glucose in amounts beyond the assimilation limit, an occa- 
sional excretion of glycuronic acid occurs with an equivalent diminution of 
the ethereal sulphates. It is possible that the substance conjugating with 
glycuronic acid is unknown and that we have the same results as though 
similar substances were introduced by mouth. As Mayer has advanced no 
direct proof of the correctness of his theory, the decision must be left for 
the future. Clinically, the question of the highest importance is whether 
the excretion of glycuronic acid is of any diagnostic value, and whether it 
is of prognostic significance in diabetes. It does not seem wise to assume 
that a patient showing an occasional increase in the glycuronic acid excre- 
tion, which cannot be accounted for by intake or increased production of 
conjugating substances, will in the future show typical diabetes. Edsall does 
not believe in the value of glycuronic acid in the diagnosis of a latent diabetes, 
nor does Neuberg regard an increased excretion of glycuronic acid as the origi- 
nal derangement which may determine other deviations from health. ^ 

^ Ztschr. f. physiol. Chem., 1901, XXXII, 518; Berl. klin. Wchnschr., 1903, XL, 292 
and 514. See Biberfeld, Biochem. Ztschr., 1914, LXV, 479; Roger and Chiray, Bull. 
I'Acad. de Med., 191 5, LXXIII, 444. 

2 See Conzen, Ztschr. f. klin. Med., 1912, LXXV, 426. 

3 See Medigreceanu (Jour. Exper. Med., 1913, XVIII, 259) for a discussion of the glycu- 
ronic acid excretion in pneumococcus infections. Roger, C. R., Soc. bioL, Paris, 1915, 



THE URINE ST,S 

Very few of the conjugated glycuronates show reducing action when 
treated with copper solutions using the precautions previously laid down. 
The chloral and camphor compounds are much more apt to produce typical 
reduction, but even these require heating for somewhat longer periods than 
does glucose. A diagnosis of a glycuronic acid excretion is based upon the 
following points : The fresh urine is levorotatory, but shows little or no reduc- 
ing properties and does not ferment. This same finding will be observed if 
j3-oxybutyric acid be present so that a diagnosis may not rest on these findings 
alone. After being boiled with dilute acid for a period varying from one- 
quarter to three-quarters of an hour, the levorotation is changed to dextroro- 
tation and the urine shows strong reducing powers. Such tests will not be 
given by jS-oxybutyric acid. In some cases after heating the urine with acid 
the action upon light may remain levorotatory or the solution may be opti- 
cally inactive, on account of the fact that the conjugating substance may be 
levorotatory or that complete hydrolysis has not been effected. On heating 
the urine for some time with Bial's modification of the orcin test a positive 
reaction appears with the liberated glycuronic acid. This acid crystallizes 
with phenylhydrazin forming distinct yellow needles which melt at 114 to 
ii5°C. This test is, however, not readily obtained so that it is difficult to 
identify glycuronic acid by its osazone. It will be seen, therefore, that gly- 
curonic acid is differentiated from the pentoses largely by its levorotation 
when in the conjugated state or dextrorotation when free. 

Tollens (Ber. d. d. chem. Ges., 1908, XLI, 1788) has advanced a test which 
permits of a clear differentiation between glycuronic acid and pentose. To 
5 c.c. of urine add J^^ c.c. of a i per cent, alcoholic solution of naphthoresorcin 
and 5 c.c. of concentrated HCl. Warm over the free flame to boiling or place 
in the boiling water-bath for 15 minutes. Allow to stand for 4 minutes and 
then cool under running water. Add an equal volume of ether and shake vig- 
orously. On separating, the ether layer shows, in the presence of glycuronic 
acid, a violet to blue coloration. Spectroscopic examination reveals a sharp 
absorption band at the D line. The test is not given by pentose. (See 
Tollens, Ztschr. f. physiol. Chem., 1908, LVI, 115; Jolles, Ibid., i9i2,LXXXI, 
203; also, Neuberg and Schewket, Biochem. Ztschr., 191 2, XLIV, 502.) 

Goldschmiedt^ has introduced a test which Mayerhofer^ believes of great 
value to the pediatrician, as it indicates intestinal derangement better than 
indican. One -half to i c.c. of urine is treated with 2 drops of a 15 per cent, 
alcoholic solution of a-naphthol. Carefully overlay 3 to 4 c.c. of concentrated 
H2SO4 with this mixture. A violet ring is observed, which changes to a dis- 
tinct emerald green on standing. In this test the urine must be free from 
nitrites and nitrates. 

LXXVIII, 714; Jean, Arch. Mens d'Obs. et de Gyn., 1915, IV, 383; Roger and Chiray, 
Gaz. Med. de Paris, 1915, LXXXVI, 46; Barbier, x\rch. d. Med. des. Enf., 1916, XIX, 225; 
Roger, Arch. d. Mai. de I'App. Dig., 1918, IX, Oi; Presse Med., 1916, XXIV, 217; Gautier, 
Ibid., 339; Pi Suner, Siglo Med., 1917, LXIV, 146; Chiray. Paris Med., 1919, IX, 359. 

^ Ztschr, f. physiol. Chem., 1910, LXV, 389; 1910, LXVII, 194. 

2 Ztschr. f. physiol. Chem., 1911, LXX, 391. 



334 DIAGNOSTIC METHODS 

Neuberg's Test.^ 

Five hundred c.c. of urine are treated with sufficient sulphuric or phos- 
phoric acid to make the acidity from i to 2 per cent. This acidified urine is 
then heated in an autoclave for two hours at a temperature of ii5°C. The 
mixture is then cooled, neutralized with sodium carbonate, acidulated with 
acetic acid, and filtered. Two hundred and fifty c.c. of the filtrate are mixed 
with a hot aqueous solution of 5 grams of parabromphenylhydrazin hydro- 
chlorate and 6 grams of sodium acetate. The mixture becomes cloudy at once, 
but on heating the cloudiness will disappear. As the mixture cools needle- 
shaped crystals will separate out and may be filtered off, the filtrate being 
again heated and cooled to obtain more crystals. This process may be re- 
peated until no more crystals form. These crystals are then washed with 
distilled water followed by absolute alcohol, and are then recrystallized by 
dissolving in 60 per cent, alcohol and gradually evaporating. They are clear 
yellow in color, melt at 236°C., and show marked levorotation when dissolved 
in a mixture of pyridin (4) and absolute alcohol (6). This test is distinctive. 
(3) Acetone Bodies. 

By the acetone bodies^ we mean acetone, diacetic acid and jS-oxybutyric 
acid. The latter of these is the mother substance so that this group would 
better be called the jS-oxybutyric bodies. The chemical relation between 
these bodies is very close, the /3-oxybutyric acid being oxidized to diacetic 
acid, which then splits up into acetone and carbon-dioxid. This may be 
seen from the following formulae : 

CH3 -CHOH -CH2 -COOH /?-oxybutyric acid. 
CH3 -CO -CH2 -COOH Diacetic acid. 

CH3 -CO -CH3 Acetone. 

Formerly these substances were supposed to be derived from protein material, 
the j8-oxybutyric acid being formed from the /5-amino acids by desamidiza- 
tion and oxidation in the (3 position. This theory is, however, not generally 
held at present, being replaced by the more modern idea that the fats are the 
chief source of the acetone bodies. It has been found that in perfectly sound, 
well-nourished individuals the addition of fat causes only a very slight in- 
crease in the output of acetone bodies and, strangely enough, that butyric 
acid itself causes no acetonuria. On the other hand in normal individuals 
from whom the dietary carbohydrate has been removed or in a diabetic who 
is not utilizing what carbohydrate he may be allowed, a marked excretion of 
acetone bodies may occur. While a portion of this acetone may possibly be 
derived from the carbohydrate groups of the protein molecules, it can hardly 
explain the enormous excretion in diabetes, as the amount of protein cata- 
boUsm, as shown by the urinary nitrogen, is greatly insufficient to yield any 
such amount of acetone bodies. The fats are, therefore, the more probable 

1 Ber. d. d. chem. Ges., 1910, XXXII, 2395; Ztschr. f. physiol. chem., 1905, XLIV, 127. 

2 See Waldvogel, Die Acetonkorper, Stuttgart, 1903; Magnus-Levy, Ergebnisse der klin. 
Med., Jena, i9o8;Lepine, Rev. de Med., 1913, XXXIII, 601; Marriott, Jour. Biol. Chem., 
1914, XVIII, 241. 



THE URINE 335 

source of these bodies. As long as the body is suppHed sufficient carbohydrate 
or is able to oxidize a sufficient amount the acetone bodies of the urine remain 
low; but when the system is no longer capable of oxidizing the carbohydrates, 
the amount of acetone bodies increases to a marked extent. We see, there- 
fore, that the older method of allowing diabetics practically no carbohydrate 
food may directly lead to aggravation of the symptoms which the withdrawal 
was supposed to remedy. In other words, the normal or the diabetic indi- 
vidual must have a certain amount of carbohydrate food in order that proper 
metabolism may be maintained.^ This is not the time or place to discuss the 
therapy or dietetic treatment of diabetes, but it must be remembered that the 
most successful diet is one which contains carbohydrates up to the point of 
tolerance. Even here we find that certain types of carbohydrates may be 
given to diabetics without increasing the glycosuria, while at the same time 
leading to a diminution in the excretion of acetone bodies. 

The condition arising from a surcharging of the blood with these acetone 
bodies is known as acidosis ^ (This definition applies especially to diabetes 
and has no reference to the acidosis of other types. A better word for this 
condition is ketosis.) For a long time it was supposed that the carbohy- 
drates were not only accountable for glycosuria, but also for the acetonuria 
and acidosis noted in diabetes. In the advance of pathologic chemistry it 
has been shown that, instead of causing these latter symptoms and conditions, 
the carbohydrates in reality lessened them. This may be shown by the 
administration of a definite amount of sugar, especially in the milder types of 
diabetes, to patients from whose diet sugars have been previously excluded. 
The omission of sugar from the diet forces the organism to utilize its protein 
and fat and thus gives rise to an accumulation of nitrogenous and fatty meta- 
bolic products as well as to an increase in the acids of the body fluids. In this 
way an acidosis already present would be increased in intensity. If the carbo- 
hydrate-free diet be continued for some time, a readjustment takes place and 
the acetonuria may gradually diminish, as is instanced by the fact that certain 
races show no acetonuria even though on an absolutely carbohydrate-free diet. 

With regard to the proteins as the mother substances of these bodies, we 
must admit that their influence is to some extent a double one.^ In the first 
place protein tends to diminish the acetonuria on account of its carbohydrate 
content, those proteins containing the greatest number of carbohydrate 
groups not necessarily exerting the greatest effect either on this condition or on 
the glycosuria. With a diet excessive in protein the influence is, however, 
not of this sort. The sulphuric and phosphoric acids as well as the small 
amount of acetone bodies formed by the hydrolysis of the protein tend to in- 
crease an existing acidosis, while the carbohydrates formed in the splitting of 

1 See Blodgett, New York, Med. Jour., 191 5, CII, 458. 

^Folin and Denis, Jour. Biol. Chem., 1915, XXI, 183; Stern, Arch. Diag., 1915, VIII, 
128; Beddard, Pembrey and Spriggs, Brit. Med. Jour., 1915, II, 389. See Straub, Deutsch. 
Arch. f. klin. Med., 1913, CIX, 223; also, Benedict and Joslin, Deutsch. Arch. f. klin. Med., 
1913, CXI, 333. For a discussion of the newer conceptions of acidosis and of the methods 
of estimating the degree of acidosis see section on Blood. 

3 See Pauly and Boulud, Lyon Med., 191 7, CXXVI, 118. 



S3^ DIAGNOSTIC METHODS 

these proteins may greatly increase an existing glycosuria. These points, 
together with the fact that the products of nitrogenous metabolism may 
greatly increase the osmotic tension of the blood and thus lead to disordered 
cell function, show us that proteins cannot be advantageous as an exclusive diet 
in diabetes. As is well known, the nitrogenous excretion is much more marked 
in a diabetic than in a nondiabetic owing to several factors. In the first 
place, the diabetic consumes more protein than the normal individual be- 
cause his diet is limited as regards carbohydrates and must be made up to a req- 
uisite caloric value by protein and fat. Secondly, owing to the lack of the 
protein-sparing function of the carbohydrates, excessive protein is broken 
down and elaborated in order to furnish a portion of the energy necessary to 
maintain the body function. It must, however, be said that the diabetic 
protects himself for a time from the unusual loss of protein by the utili- 
zation of fat. 

Concerning the fats, it is to be recalled that, although formerly accredited 
with no power of influencing acetonuria, to-day they are regarded as directly 
affecting this condition to a great extent. This is true of the fatty acids, 
especially of the lower members, and not of the neutral fats. If the conten- 
tion of Kastle and Loevenhart be true, that a reversible action of lipase con- 
verts the fatty acids and glycerin formed by a previous hydrolysis again into 
neutral fats, then the influence of fats on the acidosis is variable, or else we 
must assume a lack of lipase in the cells of the diabetic. We know that the 
fatty acids belong to the ketoplastic group (substances increasing excretion 
of acetone bodies), yet as Borchardt has recently shown this ketoplastic 
action is, doubtless, due to the union of the fatty acids with glycerin, thus 
withdrawing from the system the antiketoplastic body, glycerin, and ena- 
bling the remaining fatty acids to exert their influence on the formation of the 
acetone bodies. Fats do not increase an existing glycosuria as many experi- 
ments have shown, yet we must grant that a formation of sugar from fat 
does take place. Von Noorden speaks of a ''facultative formation of sugar 
from fat," referring to the fact that the demand for sugar may become so 
great that this source is called upon to furnish its quota of carbohydrates. 
We must also remember that the synthesis of fat from disintegrated carbo- 
hydrate is much affected in diabetes. Were this not the case, a large part 
of the sugar, reaching the blood as such, would be synthesized by the fat- 
forming cells and glycosuria would be diminished. Conceiving this latter 
function to be normal while the former is abnormal, we may readily see the 
close relationship between obesity and later diabetes." With an excessive diet 
of fat no more fat is oxidized than when the diet is low in fat. In the latter 
case, the body-fat is utilized to furnish the difference, while in the former the 
excess is deposited in the usual fat depositories. 

Besides the excretion of acetone bodies observed in diabetes, we find 
fever, carcinoma, inanition, lesions of the central nervous system, digestive 
disturbances, delayed chloroform poisoning, cases of pregnancy in which 
death^of the fetus has occurred or in which persistent toxic vomiting is noted, 
cyclic vomiting, and other conditions associated with an increased output of 



THE URINE 337 

the acetone bodies.^ According to Mohr, most of the cases may be traceable 
either to limitation of carbohydrates in the food or to diminished power of 
utilizing them. Increased protein catabolispi may play a role in the patho- 
genesis of this condition. A general statement should be that the excretion of 
the acetone bodies is little influenced by the amount of fat in the food of the 
normal individual, providing the carbohydrate content of the diet is good; but 
in pathologic conditions fats play a much greater role than do proteins in 
bringing about an acetonuria. 

Asi3-oxybutyricacid is the mother substance from which the other acetone 
bodies are formed by oxidation, we should expect to find, as we actually do, 
the severest cases showing large amounts of jS-oxybutyric acid and small or 
even no excretion of the other members of this group. As a rule, it may be 
said that the more acetone the less j8-oxybutyric acid but this is not always 
the case. In the diabetic coma we usually find large amounts of the first two 
members of this group while acetone may be absolutely lacking, and, on the 
other hand, we find in some of the milder types of diabetes acetone and no 
diaceticor oxybutyricacids.2 D akin and Wakeman and Dakin^ have shown 
that the liver possesses two ferments by which the mutual interconversion of 
i3-oxybutyric acid and aceto-acetic acid may be effected, the one being an 
oxidizing process, the other a reduction process. The acidosis may, therefore, 
be more a result of the latter than of the former action. 

{a) Acetone (CH3-CO-CH3). 

Chemically, acetone is dimethyl ketone. It shows, therefore, the reac- 
tions for this group of chemical compounds but is easily confused, both with 
the aldehyds and alcohols. The urine rarely shows typical reactions for ace- 
tone if the older tests are applied directly to the urine, so that it is necessary to 
distill and examine the distillate. In this process diacetic acid is split up into 
acetone and carbon dioxid, so that it is impossible to tell whether acetone was 
preformed or was produced by heating. From the clinical standpoint it is a 
matter of indifference, as acetone and diacetic acid are so closely related that 
their clinical significance is the same, acetone representing merely a further 
stage in the oxidation of this group of bodies. In these tests the urine must 
be perfectly fresh. If it is desired to eliminate the influence of the diacetic 
acid upon the acetone reaction, the urine may be alkalinized with sodium hy- 
drate and extracted with pure ether. The ether removes the diacetic acid salt. 

^ See Fischler and Kossow, Deutsch. Arch. f. klin. Med., 1913, CXI, 479; Czapski, Arch, 
f. exper. Path. u. Pharmakol., 1914, LXXVII, 218; Zade, Arch. f. Kinderh., 1914, LXIII, i; 
Kerley, Am. Jour. Dis. Child., 1914, VIII, 292; Peabody, Arch. Int. Med., 1914, XIV, 236; 
Roily, Ztschr. f . klin. Med., 1914, LXXIX, 548; Bradner and Reimann, Am. Jour. Med. Sc, 
1915, CL, 727; Rosenbloom, Jour. A. M. A., 1915, LXV, 1715; Rowland and Marriott, 
Bull. Johns Hopk. Hosp., 1916, XXVII, 63; Woodyatt, Jour. A. M. A., 1916, LXVI, 1910; 
Chapin and Pease, Ibid., LXVII, 1351; Mills and Wearne, Psych. Bull., 1916, IX, 413; 
Thomson, Edinburgh Med. Jour., 191 6, XVII, 298; Higgins, Peabody, and Fitz, Jour. Med. 
Res., 1916, XXXIV, 263; Veeder and Johnston, Am. Jour. Dis. Child., 1916, XI, 291; How- 
land and Marriott, Ibid., 309; Ibid., XII, 459; Veeder and Johnston, Ibid., 1917, XIII, 
89; Lackner and Gauss, Ibid., 209; Schloss and Stetson, Ibid., 218; Means and Rogers, 
Am. Jour. Med. Sc, 1917, CLIII, 420; Weber, Brit. Jour. Child. Dis., 1917, XIV, 33. 

2 See McCaskey, Jour. A. M. A., 191 6, LXVI, 350. 

3 Jour. Biol. Chem., 1910, VIII, 97 Ibid., 1910, VIII, 105; Fittipaldi, Gaz. d. Osp., 
191 7, XXXVIII, 99. 



33^ DIAGNOSTIC METHODS 

Legal's Test (Le Nobel's Test). 
To a few ex. of the urine are added a few drops of a fairly concentrated 
solution of sodium nitroprussid and then sodium or potassium hydrate until 
the mixture is strongly alkaline. A ruby-red color, later changing to yellow, 
appears in the presence of acetone. It will be remembered that this same test 
is given by creatinin, so that further modifications are necessary to permit of 
differentiation. If the ruby-red solution be treated with an excess of glacial 
acetic acid, the first red color will change into a carmine or reddish-purple 
color in the presence of acetone, while the same treatment with creatinin 
solutions yields a yellow, changing to green and finally to a blue coloration. 
As Le Nobel has found, ammonium hydrate does not give this reaction with 
creatinin, but with acetone, although the reaction is much slower in appear- 
ing. This test is given by diacetic acid, by alcohol, and by acetic aldehyd, so 
that it is not especially distinctive for acetone. It is perhaps better if this 
test is to be used at all that the urine be previously acidified and distilled, the 
distillate yielding a reaction which is more sensitive, according to Studer, 
although diacetic acid will thus be converted into acetone. 

Lange^ has modified this test as follows: To the suspected urine add H 
c.c. of glacial acetic acid and a few drops of a freshly prepared aqueous solu- 
tion of sodium nitroprussid. Mix thoroughly and overlay the mixture with 2 
c.c. of concentrated ammonium hydroxid. At the point of contact a pur- 
pUsh-red ring is observed in the presence of acetone. 

Lieben's Test. 

To a few c.c. of urine^ or, preferably, of the distillate are added a few drops 
of concentrated sodium or potassium hydrate and a few drops of a solution of 
iodin in potassium iodid. On slightly warming the mixture yellow crystals 
of iodoform will separate, which may be recognized by their characteristic 
odor as well as by their hexagonal shape when examined under the microscope. 
This test is given by alcohol as well as by aldehyds, and will show amounts of 
acetone varying between J^oo and Ho 00 of 3, mg. 

Guiming's Test. 

This is a modification of the previous test and is much more specific, being 
given only by acetone. To the distillate from the urine are added a few drops 
of an alcoholic solution of iodin and the mixture treated with ammonia until a 
black precipitate of nitrogen iodid forms. On allowing the tube to stand for 
periods varying between 12 and 24 hours, this black precipitate disappears, 
leaving a yellow sediment of iodoform, which may be recognized as mentioned 
above. This test is less delicate than the original one of Lieben, detecting 
acetone when present in amounts oi}/ioo ois, mg. per c.c. of urine. 

1 Miinch. med. Wchnschr., 1906, LIII,i764. See, also, Yanagawa, Biochem. Ztschr., 
1914, LXI, 256. 

2 Bardach (Ztschr. f. physiol. Chem., 1908, LIV, 355) and, more recently, Rosenbloom 
(Jour. Am. Med. Assn., 191 2, LIX, 445) have shown that protein and many of its deriva- 
tives prevent the formation of the characteristic iodoform crystals. Hence the distillate 
should always be used. 



THE URINE 339 

Frommer's Test. 

Frommer^ has introduced a test which seems to be distinctive for acetone 
and at the same time very delicate. It is based upon the fact that acetone 
reacts with saUcyl aldehyd to form dioxydibenzoylacetone, according to the 
following equations. The alkali salt is distinctly red. 

C6H4OHCHO+ (CHs) 2CO -= C6H4OHCH = CHCH3CO+H2O 
C6H40HCH = CH+CHOOHC6H4 = C6H40HCH = CH +H2O 

CH3 / 

CH = CHOHC6H4 

The test may be performed as follows: Ten c.c. of urine are strongly 
alkalinized with potassium hydrate and 10 to 12 drops of a 10 per cent, solu- 
tion of saUcyl aldehyd in absolute alcohol are added and the mixture warmed 
to about 7o°C. In the presence of acetone the fluid becomes yellow, then red, 
later purplish-red, and, on long standing, dark red. In the absence of acetone 
the color of the urine is practically unchanged. This test is said by Frommer 
to indicate the presence of one one-millionth of a gram in 8 c.c. of urine. 

Instead of applying the test as above, we may add about i gram of po- 
tassium hydrate (in the sohd state) to 10 c.c. of urine and, without waiting for 
complete solution to occur, treat the mixture with 10 to 12 drops of the alco- 
hohc solution of salicyl aldehyd and warm to 70°. i\t the zone of contact of 
the alkah and the salicyl aldehyd an intense purplish-red ring is observed 
only in the presence of acetone. 

The WTiter has frequently used this test and finds it very satisfactory, as 
it does not react with diacetic acid unless the heating be long continued. 

Quantitative Determination of Acetone. 

Many of the quantitative methods given for acetone are more or less com- 
pHcated and at the same time do not yield absolutely accurate results. The 
writer selects, therefore, those proving most satisfactory in his hands. All of 
these methods, with the exception of Folin's, give the amount of preformed 
acetone as well as that derived, by distillation, from the diacetic acid.^ 

Huppert-Messinger Method. 

The principle of this method is the determination of the amount of iodin 
necessary to transform into iodoform the acetone derived in the distillation of 
the urine. Knowing this factor, a simple calculation yields the amount of 
acetone present. 

For this determination certain solutions are necessary: 
(i) A 50 per cent, solution of acetic acid. 

(2) A tenth-normal solution of sodium thiosulphate. In preparing 
this solution 24.8 grams of crystalhzed sodium thiosulphate (Na2S2035H20) 

1 Berl. klin. Wchnschr., 1905, XLII, 1008. See Engfeldt, Ibid., 1915, LII, 458; Csonka 
(Jour. Biol. Chem., 1916, XXVII, 209) has introduced a colorimetric method for determina- 
tion of acetone, based on this test, which is reliable and accurate but is not discussed here 
as the salicyl aldehyd is practically unobtainable. 

2 See Sammet, Ztschr. f. physiol. Chem., 1913. LXXXIII, 212; Lenk, Biochem. Ztschr., 
1916, LXXV, 224. 



340 DIAGNOSTIC METHODS 

are carefully weighed out and dissolved in distilled water, the solution being 
made in a volumetric flask and diluted exactly to the liter mark. 

(3) A tenth-normal solution of iodin. This solution requires exactly 
12.685 grams of iodin in one hter. As iodin is difficultly weighable on ac- 
count of its volatility, it is advisable to weigh out approximately 13 grams of 
iodin and dissolve in approximately i liter of water to which has been added 
25 grams of potassium iodid. This solution is then standardized by titrating 
against the previously made tenth-normal thiosulphate solution, using thin 
starch paste as an indicator and adding the iodin solution from a buret until 
the blue color of iodid of starch just appears. This determination is then 
confirmed by duplicate estimations. Twenty c.c. of the iodin solution should 
be the equivalent of 20 c.c. of the thiosulphate solution, so that the necessary 
dilution of the iodin solution may be determined by the formula previously 
given under Determination of Chlorids in the Urine (page 202). One c.c. 
of the standard tenth-normal iodin solution equals 0.012685 gram of iodin 
and represents 0.967 mg. of acetone. 

Technic. 

Five hundred c.c. of normal acid urine or 100 c.c. of acetone-rich urine are 
treated with 2 c.c. of 50 per cent, acetic acid for every 100 c.c. of urine and 
distilled until %o of the volume has passed over. The distillate is collected 
in a receiving flask, which is cooled with ice and which contains water to ab- 
sorb the acetone. The flask is tightly closed with a doubly perforated stopper^ 
through one hole of which passes the tube of the condenser reaching below the 
surface of the water and through the second opening a bulb filled with water 
to act as a safety bulb. The tube is then washed with distilled water and the 
fluid in the safety bulb is emptied into the receiving flask. It is important 
that the distilling flask be disconnected before the heat is shut off, as other- 
wise the fluid might suck back. This distillate is treated with calcium car- 
bonate to remove any nitrous or formic acid which may have distilled over, and 
the mixture thoroughly shaken. 

This distillate is then acidified by the addition of i c.c. of dilute sulphuric 
acid (diluted eight times) and redistilled until one-tenth of the volume re- 
mains in the distilling flask. This second distillate is received in a flask 
arranged as in the previous distillation. It is then poured into a flask, which 
can be closed with a tight-fitting glass stopper. Distillate and wash-water 
must not fill the vessel more than one-third full. A large excess of carefully 
measured N/io iodin solution is added, the mixture weU shaken, and strong 
sodium hydrate solution added drop by drop. The flask is then stoppered, 
shaken for one-fourth minute, and allowed to stand for five minutes. The 
stopper is then removed, the fluid clinging to it washed into the flask, and the 
fluid acidified with strong HCl. The excess of iodin is then determined by 
allowing N/io thiosulphate solution to flow from a buret until the mixture 
is but slightly yellow, when a few c.c. of starch paste are added and the 
titration continued until the blue color just disappears. One c.c. of iodin 
solution used by the fluid corresponds to 0.967 mg. acetone, so that all that 
is necessary is to subtract the number of c.c. of thiosulphate used from the 



THE URINE 341 

number of c.c. of iodin solution added and multiply by the above factor. 
The result is the acetone in the amount of urine taken. 

This method yields results which are from 5 to 10 per cent, low,^ so that 
slight variations in the acetone excretion, as determined by this method, have 
it tie significance. 

Folin's Method. 
This method^ yields only the preformed acetone occurring in the urine and 
does not regard the acetone which may be derived from the diacetic acid. 
If the urine be distilled and this method employed, the total possible acetone 
will result. It is somewhat more accurate than the previous method and is 
much more simple and less time-consuming. The writer finds it extremely 
serviceable and adopts it in all acetone determinations. 

Twenty-five c.c. of urine are measured into an aerometer cylinder, similar 
to that used in Folin's ammonia apparatus, a few drops of 10 per cent, 
phosphoric acid, 10 grams of sodium chlorid, and a little petroleum are added. 
In the absorbing bottle which is fitted with an absorption bulb are placed 300 
c.c. of water, 20 c.c. of 40 per cent, potassium hydrate, and an excess of 
tenth-normal iodin solution. The apparatus is connected with the filter- 
pump in the same manner as described under Ammonia and an air-current 
drawn through for one-half hour. The air should not be passed as rapidly 
as in the ammonia determination. Each worker should check his air blast 
by control estimations, using known solutions of acetone. In this way the 
acetone will be removed from the urine and converted, in the receiving flask, 
into iodoform. The contents of the receiving flask are acidified with concen- 
trated HCl, using 10 c.c. of acid for every 10 c.c. of alkaU previously used, and 
the excess of iodin titrated with tenth-normal sodium thiosulphate as in the 
previous method. 

If this method be employed with the distilled urine, subtract from the 
total acetone, obtained in the distillate, the preformed acetone, derived from 
the fresh urine, and obtain the acetone referable to diacetic acid. If this 
value be multiplied by 1.758 the result will be the amount of diacetic acid, as 
such, present in the urine taken. 

Shaffer has recently introduced a method for the determination of the 
total acetone, which is closely related to his method for estimating /3-oxybu- 
tyric acid and will be discussed in a later section.^ 

{b) Diacetic acid (CH3-CO-CH2-COOH). 

This substance is derived from |S-oxybutyric acid and is the precursor of 
acetone. As a rule, if acetone be present in large amounts, diacetic acid is 
also present and indicates a much graver condition than does the mere pres- 
ence of acetone. If Folin's contentions are true, most of what we now call 
acetone is, in reality, diacetic acid, so that this latter substance should be 
tested for in routine urinary work. The remarks previously made regarding 

^See Marriott, Jour. Biol. Chem., 1913, XVI, 281. 

2 Jour. Biol. Chem., 1907, III, 177; also, Folin and Denis, Ibid., 1914, XVIII, 263. 

3 See Issoglio, Giorn. farm, chim., I1919, LXVI, 301 and Jour. Chem. Soc, 1919, CXVI, 
304, for a nephelometric method. 



342 DIAGNOSTIC METHODS 

the appearance of acetone in the urine also hold for diacetic acid.^ This 
substance is very volatile and disappears from the urine in a relatively short 
time so that the tests should be applied to perfectly fresh specimens. 

Gerhardt's Test. 

To lo c.c. of urine are added a few drops of lo per cent, ferric chlorid 
solution. This is best added as long as a precipitate of phosphates occurs, 
these latter bodies being then filtered off. To the filtrate are added a few 
drops more of ferric chlorid solution when the urine shows a Bordeaux-red 
color in the presence of diacetic acid. This color appears cherry-red by 
transmitted light and purplish-red by reflected light. 

Unfortunately, however, this test is not specific for diacetic acid.^ A 
red color is observed in the presence of cyanates, formates, acetates, phenol 
compounds, salicylates, the conjugated glycuronates of phenacetin, antipy- 
rin, thalhn, kryofin, and kairin, as well as meconic acid which may be excreted 
after intake of opium. If the urine be heated, diacetic acid decomposes more 
or less completely into acetone and carbon dioxid, so that the red color dae to 
diacetic acid will either disappear or become much weaker, while that with 
the other substances mentioned is not affected by heat. This same disap- 
pearance of color is noted if the mixture be allowed to stand from 24 to 48 
hours. Schreiber recommends filtration of the urine through animal char- 
coal, using about 10 grams of charcoal to 100 c.c. of urine. Antipyrin, phe- 
nacetin, and kryofin are retained while sufficient of the diacetic acid passes 
through to permit of detection. The urine may even be acidified with 
sulphuric acid and extracted with ether. This ethereal solution, which con- 
tains the diacetic acid, is then shaken with water and ferric chlorid solution 
added. A deep red color will be seen in the watery layer in the presence of 
diacetic acid. If the red color, on addition of ferric chlorid, be due to the 
presence of meconic acid, it will disappear on the further addition of stannous 
chlorid or of alkali hypochlorites, while that due to diacetic acid is unaffected. 
This test is, perhaps, more frequently used than any other test for diacetic 
acid in the urine, but is by no means as serviceable as the following. 

Arnold's Test. 

For the performance of this test two reagents are employed: (i) a solution 
consisting of i gram of paraamidoacetophenon, 100 c.c. of distilled water, 
and 2 c.c. of concentrated hydrochloric acid; (2) i per cent, sodium nitrite 
solution. Fifteen c.c. of urine are treated with a mixture consisting of 10 
c.c. of solution i and 5 c.c. of solution 2 and one drop of concentrated 
ammonia added. In practically all urine, whether it contains diacetic acid 
or not, a brownish-red coloration is observed which changes in the absence 
of diacetic acid into yellow if the mixture be treated with an excess of concen- 
trated hydrochloric acid, while if diacetic acid be present the color changes 

1 See Dakin and Dudley, Jour. Biol. Chem., 1913, XVI, 515. 

2 See Steensma and Koopman, Nederl. Tijdschr. v. Geneesk., 1914, LVIII, 800; Licht- 
witz, Berl. klin. Wchnschr., 1915, LII, 399; Barret, Jour. Lab. and Clin. Med., 191 7, II, 
203; Mitchell (Jour, pharm. chim., 1919, XX, 31) modifies this test by using ferric alum 
instead of chlorid. 



THE URINE 343 

to a beautiful purple on the addition of acid. If the mixture be shaken the 
foam also shows a distinct violet coloration. 

As this test is somewhat difficult in the presence of small amounts of 
diacetic acid, Lipliawsky^ has modified it as follows: Six ex. of solution i 
and 3 c.c. of solution 2 are treated with the same volume of urine, a drop of 
ammonia is added and the mixture shaken, when it assumes a brick-red 
color. According to the probable contents of the urine in diacetic acid 10 
drops to 2 c.c. of this mixture are treated with 15 to 20 c.c. of concentrated 
hydrochloric acid, 3 c.c. of chloroform and two to four drops of ferric chlorid 
solution. The test-tube is then closed with a cork and gently shaken for one- 
half to one minute. In the presence of traces of diacetic acid the chloro- 
form assumes a characteristic violet coloration, while in the absence of this 
acid the color is yellow or light red. This test is positive for one part in 
400,000 of water. Acetone and /3-oxybutyric acid do not react with this 
test nor do the drugs previously mentioned under the Gerhardt Test interfere. 
If the urine is highly colored it is advisable to filter through animal charcoal. 

(c) ^-OxybutyricAcid^ (CH3-CHOH-CH2-COOH). 

This acid, the mother substance of the acetone bodies, is found in the 
urine in extreme cases of the conditions described under Acetone. The 
amount excreted may vary from traces to as high as 100 grams. Klilz has 
reported an excretion of 225 grams in 24 hours. The free acid is practically 
never found in the urine, being excreted either as the ammonium, sodium, or 
potassium salt. These salts as weU as the free acid are levorotatory and may be 
detected by the polariscope after previous fermentation of the carbohydrates. 

In the condition known as diabetic coma a specific intoxication with 
|8-oxybutyric acid is assumed as the causative factor. While this is undoubt- 
edly true to a large extent it cannot be regarded as the only factor in diabetic 
coma, as administration of j8-oxybutyric acid in large quantities will not neces- 
sarily lead to such a syndrome unless largely retained. We do undoubtedly 
have an acidosis which acts by ultimately depriving the tissues of fixed alka- 
lies and may be regarded, therefore, as of great importance in following a 
diabetic case. Were this acid, per se, accountable for the entire symptoma- 
tology, we should be able, by administering alkalies, to overcome the 
effect of the acid intoxication. In some cases such therapy is extremely 
beneficial," while in others it is practically useless, as it cannot influence the 
formation of the toxic bodies and may not increase their elimination. 

As previously stated, the ammonia output of the urine is an invaluable 
guide in following the course of an acidosis, especially in diabetes. One 
gram of ammonia (NH3) is equivalent to 6.12 grams of j8-oxybutyric acid, 
especially when in excess of the amount directly due to the food. 

To detect /3-oxybutyric acid follow the method of Hart.^ To 20 c.c. of 
urine add 20 c.c. of water and a few drops of acetic acid and boil until the 
volume is reduced to about 10 c.c. (to remove acetone and diacetic acid). 

^ Deutsch. med. Wchnschr., 1901, XXVII, 151. 

2 The more scientific name for this body is &-hydroxybutyric acid. See Sassa, Biochem. 
Ztschr., 1914, LIX, 362; Kennaway, Biochem, Jour., 1914, VIII, 230 and 355. 

3 Am. Jour. Med. Sc, 1909, CXXXVII, 869. 



344 DIAGNOSTIC METHODS 

To the residue add sufficient water to make the volume 20 c.c. and place 10 
c.c. in each of two test-tubes. To one of these add i c.c. of hydrogen 
peroxid, warm gently and allow to cool. Apply Lange's test for acetone 
(see p. s^S) and allow the tubes to stand for a few hours. No reaction is 
observed in the tube which contains no hydrogen peroxid, while a distinct 
red zone is seen in the other. 

Quantitative Determination. 

A large number of methods have been advanced for the determination 
of jS-oxybutyric acid in the urine. Among these we find the methods of Kiilz, 
Tollens, Wolpe, Magnus-Levy, Bergell, Stadelmann, and Darmstadter. 
While some of these are accurate under certain conditions, so many precau- 
tions must be taken that widely varying results may be obtained. On the 
other hand, the method of Magnus-Levy, which is undoubtedly very exact, 
requires 24 hours, while that of Bergell depends to a large extent upon the 
condition of the powder which is extracted with ether. 
Black's Method. 

This method^ is a modification of those of Magnus-Levy and of Bergell, 
but in the writer's hands has given much more satisfactory results. One 
hundred c.c. of urine are faintly alkalinized with sodium carbonate and evapo- 
rated in a porcelain dish to one-third or one-fourth of the original volume. 
The residue is then concentrated to about 10 c.c. on a water bath in order 
completely to remove the diacetic acid. ■ This is then cooled, acidified with 
a few drops of hydrochloric acid, and made into a thick paste with plaster 
of Paris. This mixture soon begins to set, when it is stirred and broken up 
with a glass rod. This porous mass is then transferred to a Soxhlet apparatus 
and extracted with pure ether for two hours. The ether extract is evapo- 
rated, the residue taken up with water, decolorized with bone-black and fil- 
tered perfectly clear. The filtrate is then made up to 25 c.c. and its 
rotation determined with a polariscope. In determining the amount of jS-oxy- 
butyric acid from its rotation we must make use of the following calcula- 
tion. The specific rotation of the free acid is — 24.12 in a decimeter tube. 
One division of the scale in the case of glucose equals 2 per cent., so that we 
may find the percentage of jS-oxybutyric acid by the following proportion: 

2 : X :: 24.12 : 52.7. X = 4.37 per cent. 
By a similar proportion it may be found that one division of the scale corre- 
sponds to 7.1 per cent, of sodium /3-oxybutyrate, whose specific rotation is 

-14.35. 

This method is just as exact as those of Magnus-Levy and Bergell and 
has the advantages that it is simpler, more reliable, and may be performed 
in shorter time. The greatest difficulty with this method arises in the deter- 
mination of the exact point of the scale at which the two portions of the 
polarimetric field are equally illuminated. Magnus-Levy has shown that 
a difference of J^q of 1° in reading the polariscope amounts to about three 

1 Jour. Biol. Chem., 1908, V, 207. See Van Slyke (Jour. Biol. Chem., 1917, XXXII, 
455) and Van Slyke and Fitz (Ibid., 495) for methods of determining the three acetone 
bodies. 



THE URINE 345 

grams per liter of /?-oxybutyric acid, so that great care must be used with the 
polariscope in this method as well as in all others in which it is applied. 
Shaffer's Method. 

This method^ seems to the writer to be the most desirable one that has 
been advanced. Shaffer and Marriott^ show that this method yields uni- 
formly about 90 per cent, of theoretical values. The results obtained 
must, therefore, be corrected by the addition of 10 per cent, of the amount 
found. See, also, Pribram^ in this connection. 

The principle of the method is the oxidation of jS-oxybutyric acid to ace- 
tone and carbon dioxid and the determination of the amount of acetone thus 
evolved. The acetone and diacetic acid already existing as such in the 
urine are previously determined by this method, so that it serves as a gen- 
eral one for the acetone bodies. 

From 25 to 250 c.c. of urine, depending upon the amount of j8-oxybutyric 
acid expected, are measured into a 500 c.c. volumetric flask and an excess of 
basic lead acetate and 10 c.c. of concentrated ammonia are added. In se- 
lecting the amount of urine to be taken one should use sufficient to yield from 
25 to 50 mg. of acetone derived from /3-oxybutyric acid. The solution in the 
flask is then diluted to the graduating mark, is thoroughly shaken, and filtered. 
Two hundred c.c. of the filtrate (representing % of the original volume of 
urine taken) are diluted with water to 500 or 600 c.c. 15 c.c. of concentrated 
sulphuric acid and a few grams of talcum are added, and the mixture distilled 
until 200 to 250 c.c. of distillate collects (distillate A). 

In this distillation, the distilling flask, which may be an 800 c.c. Kjeldahl 
flask, should be fitted with a dropping funnel and water run in to prevent 
the volume of fluid in the flask from becoming less than 400 c.c. This 
distillate (A) contains the preformed acetone and that from the diacetic acid 
as well as volatile fatty acids which may be present in the urine. To remove 
the fatty acids, especially formic acid, the distillate A is redistilled after adding 
5 c.c. of ID per cent, sodium hydrate solution. This distillate (^2) is then 
titrated with standard tenth-normal iodin and thiosulphate solutions as 
described in the Huppert-Messinger method for determination of acetone. 

The residue of urine and sulphuric acid from which A was obtained is again 
distilled, dropping in 400 to 600 c.c. of o.i per cent, to 0.5 per cent, potassium 
bichromate^ solution. This bichromate must not be added faster than the 
distillate collects unless the boiling liquid turns a pure green color, indicating 
that the bichromate is being used up more rapidly. When about 500 c.c. of 
distillate (B) have collected, 20 c.c. of 3 per cent. H2O2 are added to the dis- 

1 Jour. Biol. Chem., 1908, V, 211. 

2 Jour. Biol. Chem., 1913, XVI, 265. Folin and Denis (Jour. Biol. Chem., 1914, XVIII, 
263) show that their turbidity method, using small amounts of urine, yields theoretical 
amounts of acetone. 

^ Ztschr. f. exp. Path, u Therap., 191 2, X, 279 and 284. 

^ Cooke and Gorslin (Jour. Biol. Chem., 191 1, X, 291) have wisely advised the following 
modification. The residue^of urine and sulphuric acid is diluted to 600 c.c. and 5 c.c. of a 5 
per cent, solution of potassium bichromate are added. DistUl down to 300 c.c. and add 
water through the dropping tube to 500 c.c. Distill until 500 c.c. have been obtained. Ad- 
ditional 5 per cent, bichromate is run in, a few cubic centimeters at a time, whenever the 
distilling solution shows a greenish tinge. 



346 DIAGNOSTltSMETHODS 

tillate^ together with a few c.c. of sodium hydrate solution and this redistilled. 
This second distillate (B2) is then titrated with tenth-normal iodin and thio- 
sulphate solution. One mg. of acetone represents 1.794 mg. of j8-oxybutyric 
acid. 

(4) Abnormal Pigments. 

(a) Blood Pigments. 

The principal blood pigment appearing in the urine is hemoglobin, which 
has been previously discussed under the heading of Protein in the Urine. 
Certain derivatives of this pigment are found, however, in conditions in which 
hemoglobin does not appear (see hemoglobinuria). 
Hematoporphjrrin. 

This is an iron-free derivative of hemoglobin and appears to be present 
in minute traces in normal urine. Pathologically, it has been found in cases 
of rheumatism, phthisis, Addison's disease, pericarditis, paroxysmal hemo- 
globinuria, cirrhosis of the liver, exophthalmic goiter, croupous pneumonia, 
lead poisoning, syphilis, and many acute infectious diseases. Long-continued 
use of certain hypnotics, such as sulphonal,^ trional, and tetronal, is fre- 
quently associated with the appearance of hematoporphyrinuria. 

Urine containing hematoporphyrin is usually dark-red in color, but may 
vary from a brownish-red or port- wine color to a distinct Bordeaux-red. 
Hammarsten has shown that this color is not entirely due to the hematopor- 
phyrin, but partially to other abnormal pigments whose identity is not certain. 

In examining urine for the presence of hematoporphyrin the spectro- 
scopic method is practically the only one available. If this pigment be 
present in large amounts the urine may be directly examined with the 
spectroscope, showing the four bands of alkali hematoporphyrin discussed 
in the section on Blood. As this method is not always certain and not always 
easy of application, it would seem preferable to treat 50 c.c. of urine with 10 
c.c. of 10 per cent, sodium hydrate solution. The precipitated phosphates 
carry down the pigments. This precipitate is then treated with 10 drops of 
concentrated hydrochloric acid and 15 c.c. of absolute alcohol. The solution 
is filtered if necessary and examined with the spectroscope when the two 
absorption bands of acid hematoporphyrin will be observed. 

(b) Biliary Pigments. 

Normally, bile pigments do not occur in the human urine. As was 
previously discussed in the section on UrobiHn, biliary pigments may appear 
in the urine in conditions interfering with the passage of the bile into the 
intestine or when increased formation of biliary pigments from blood pigments 
has occurred and an associated obstruction of the bile-ducts is present. 

We see, therefore, that choluria occurs in every case in which there is 
obstruction to the outflow of bile into the intestine. Thus in catarrhal jaun- 

^ See Witzemann (Jour. Biol. Chem., 1918, XXXV, 83) for the efifect of variations in 
available alkali on the yield of acetone in this process. 

2 See Pfortner, Deutsch. med. Wchnschr., 1914, XL, 1563; Fischer, Ztschr. f.physiol. 
Chem., 1915, XCV, 34; Ibid., XCVI, 148; Schumm, Ibid., 183; Hoagland, Jour. Agricul. 
Res., 1916, VII, 41; Rous, Jour. Exper. Med., 1918, XXVIII, 645; Schumm, Ztschr. f. 
physiol. Chem., 1919, CV, 158. 



THE URINE 347 

dice, biliary calculi in the common duct, carcinoma of the liver, and cirrhosis, 
bilirubinuria is frequent. Moreover, we may also find biliary pigments aris- 
ing from purely hematogenous conditions, such as pernicious anemia, malaria, 
typhoid fever, arsenical poisoning, and yellow fever. Whether this latter 
type is not really hepatogenous in origin, as Stadelmann believes, is still un- 
settled, but it would seem more plausible to assume a primary breaking down 
of the red cells and a secondary insufficiency of the liver. 

The chief biliary pigment found in the urine is bilirubin,^ which is inter- 
mediate between hemoglobin and urobilin. On oxidation of this pigment, 
either in the system or in the methods of examination, various other pigments 
may arise. Thus we may find biliverdin, bilicyanin, bilifuscin, biliprasin, 
cholecyanin, and choletelin. In the fresh urine bilirubin is the only pigment 
noted. 

A bile-containing urine may show various shadings of color, ranging from 
a greenish-yellow, through yellowish-brown, deep brown, or greenish-brown, 
to a pure green. If the urine be shaken, a yellowish or greenish-yellow foam is 
observed, while in normal urines the foam is practically colorless. The. 
presence of an excess of urobilin may also give a brownish foam. Urine con- 
taining bile always shows the presence of nucleoalbumin along with slight 
traces of serum albumin, so that bile should be regarded as a source of 
extraneous albumin. The sediment will usually be more or less colored by 
the biliary pigment, the casts in yellow fever, for instance, being usually 
distinctly bile-stained. 

Qualitative Tests. 

A large number of tests have been advanced for the detection of biliary 
pigments in the urine and many of them are distinctly unsatisfactory. If 
large amounts of bile be present, the urine, acidified with HCl, may be shaken 
out with chloroform which dissolves the bilirubin. If the chloroform be 
evaporated, rhombic crystals with rounded edges or distinct needles of a 
brownish-red color will be observed. These crystals will give the color tests 
mentioned below. 

Smith's Test. 

This test has been described under other names, as those of Trousseau, 

Kathrein, Rosin, and Marechalt. A few c.c. of urine, acidified if necessary 

with acetic acid, are treated with a i per cent, alcoholic solution of iodin in 

such a way that the latter solution is superimposed upon the urine, forming 

a distinct line of contact. If bilirubin or other biliary pigments be present, 

a beautiful emerald-green color is observed at the point of contact. This test 

is especially recommended, but it is not very sensitive, indicating only one 

part of biliary pigment in 10,000 of urine. Certain drugs, especially antipyrin, 

may lead to the formation of a green color with this test. Thymol, if used as 

a preservative, may give rise to confusion with this and other bile tests. 

^ See van den Bergh and Snapper, Nederl. Tijdschr. v. Geneesk., 191 5, II, 469, Kiister, 
Ztschr. f. physiol. Chem., 1915, XCIV, 136; Hooper and Whipple, Jour. Exper. Med., 
1916, XXIII, 137; Am. Jour. Physiol., 1916, XI^, 332; Whipple and Hooper, Ibid., 349; 
Garnier and Magnenand, C. R. soc. biol. de Paris, 1916, LXXIX, 278; Dejager, Nederl. 
Tijdschr. v. Geneesk., 1916, II, 2271; Pharm. Zentralbl., 1917, LVIII, 442. 



348 DIAGNOSTIC METHODS 

Gmelin's Test. 

One or two c.c. of nitric acid areplaced in a test-tube and the same amount 
of urine is allowed to flow from a pipet in such a way that a distinct contact 
line is formed. If the nitric acid contains a trace of nitrous acid and the urine 
biliary pigment, a distinct green ring will be observed at the line of contact. 
In some cases, as the nitric acid oxidizes the pigment, a play of colors may be 
seen from green, through a blue, violet, and red, to a yellow. The primary 
green is the characteristic color, other colorations being occasionally due to 
pigments other than biliary. This test is supposed to indicate i part of 
bilirubin in 80,000 of urine. 

If the urine contains an excess of indican a deep-blue ring may be observed 
or the combination of thds blue with the yellow of the urine may give a green. 
In the presence of skatoxyl a violet-red ring may be noted, while various me- 
dicaments may give colorations ranging through the entire spectrum. In such 
cases it is always advisable to extract the acidified urine with chloroform and 
apply the test either to the evaporated residue or to its aqueous solution. 

This test is, perhaps, more frequently used than any of the biliary tests, 
but requires considerable experience for its proper interpretation. If the 
urine be diluted the test is somewhat more distinctive. Many modifications 
of this test have been advocated, the most serviceable being the following: 

Rosenbach's Test. 

A large quantity of urine, which has been acidified with HCl, is filtered 
several times through a thick filter-paper, which, will hold back the bile- 
stained elements of the urine. It is sometimes advisable to add a little milk of 
lime to the urine before filtering, instead of the HCl, as this will throw down 
the phosphates which will carry with them the bilIcir}Digment. If the filter- 
paper and contents be dried by pressing with a second dry filter-paper and a 
drop of yellow nitric acid allowed to fall upon it, distinct rings will be seen, 
which will be colored as in the previous test, the green one being external. 
Nakayama's Test. 

This is a modification of the older Huppert test.^ Five c.c. of acid urine 
are treated with an equal volume of 10 per cent, barium chlorid solution and 
the mixture centrifuged. The barium chlorid precipitates the phosphates 
and sulphates and carries down the biliary pigments. The supernatant 
fluid is then poured off and 2 c.c. of the following reagent are added to the 
precipitate. The reagent consists of 99 c.c. of 95 per cent, alcohol, i c.c. of 
concentrated HCl, and 0.4 gram of ferric chlorid. If this mixture of precipi- 
tate and reagent be heated to boihng, a bluish-green or a brilliant green 
solution is obtained, which becomes violet or red on the addition of nitric acid. 
This test is said to indicate one part of bilirubin in 1,200,000 parts of urine. 

Hammarsten's Test. 
As the reagent In this test we use a mixture of one part of 25 per cent. 
HNO3 and 19 parts of 25 per cent. HCl. Before use one part of this reagent 
is mixed with four parts of alcohol. A few drops of the urine are added to this 

1 See Rosenbloom, New York Med. Jour., 19 14, XCIX, 229, 



THE URINE 349 

mixture, when it assumes a green color in the presence of biliary pigments. 
If the urine be treated as in Nakayama's test and the precipitate mixed with 
I to 2 c.c. of the acid alcohol reagent and the whole centrifuged for a short 
time, a green solution is obtained if one part of bilirubin in 1,000,000 of urine 
be present. 

Bile Acids. 

The bile acids, taurocholic and glycocholic acids, are found in the urine in 
the form of their sodium salts. They may be found in small amounts in the 
same conditions in which the biliary pigments are present, but their amount is 
usually so small that they cannot be detected, as a rule, without being 
previously isolated. It seems to be fairly well established that the bile acids 
must be present to the extent of 3^ per cent, before detection in the urine is 
possible. 

As the clinical significance of these acids is the same as that of the pigment 
and as their amount is so small that the methods of isolation require a fairly 
large volume of urine, the writer will refer to works on physiologic chemistry 
for such procedures.^ 

No absolute] V reliable test is known for the detection of the bile^cids in the 
untreated urine. There are, however, a few tests which are occasionally 
given providing the bile acids be present in sufficient amount. 
Hay's Test. 

This test depends upon the reduction of the surface tension of the urine in 
the presence of the bile acids. As advocated by Beddard and Pembrey, 
a pinch of powdered sulphur is sprinkled upon the surface of urine which 
should be preferably at a temperature not over i7°C. In normal urines the 
sulphur will float upon the surface, while if the urine contains bile acids the 
sulphur may sink at once indicating one part in 10,000 or may sink only 
after a few seconds to one minute, in this latter case indicating one part in 
50,000. According to Sahli this test does not discriminate between 
biliary acids and biliary pigments, but clinically it is a matter of indiffer- 
ence which one is present. Phenol or aniline compounds lower the surface 
tension of the urine so that their presence may lead to wrong conclusions.^ 
Oliver's Test. 

This test is based upon the well-known property possessed by the bile 
acids of precipitating peptone when in acid solution. The reagent is as follows : 
Powdered peptone, 8.33 grams 

Salicylic acid, 1.12 grams 

Acetic acid, 2 drops 

Distilled water, i liter 

One to two c.c. of clear filtered urine are placed in a test-tube and treated 
with 5 c.c. of the reagent. In the presence of bile acids a decided milkiness 
appears at once, being the more intense the larger the amount of bile acids. 

^ See Wieland and Sorge, Ztschr. f. physiol. Chem., 1916, XCVII, i; Jour. Chem. Soc.» 
1916, CXI, 710; Foster and Hooper, Jour. Biol. Chem., 1919, XXXVIII, 355, 367, 379, 
393, 413 and 421. 

2 See AUen (Jour. Biol. Chem., 1915, XXII, 505) for a method of measuring accurately 
the variation in surface tension of the urine. 



350 DIAGNOSTIC METHODS 

Other tests, such as those of Pettenkofer and Udranzky, are only service- 
able in the testing of the isolated bile acids. 

(c) Melanin. 

In cases of melanotic tumors the urine not infrequently contains a chro- 
mogen (melanogen), which is converted into melanin on allowing the urine 
to stand or after adding alkalies or oxidizing agents. The urine when freshly 
voided is normal in color, but on exposure to air gradually darkens until it 
becomes distinctly black. This coloration is first noticed in the upper portion 
of the urine and gradually extends toward the bottom. If ferric chlorid be 
added to the urine the coloration may be somewhat intensified so that one 
may assume an excess of phenol derivatives. It is to be remembered that 
indican must first be split up with acid before giving the coloration with 
ferric chlorid; moreover, melanin is insoluble in chloroform while the indigo is 
readily soluble. (See Eppinger.^) 

The addition of the ferric chlorid may produce a black precipitate which 
is soluble in sodium carbonate solution, from which it may be reprecipitated 
by mineral acids. This pigment may also be found in some cases of chronic 
malaria^ so that it is not absolutely pathogonomonic of melanotic tumors. 

(d) Phenol Derivatives. 

As previously mentioned in the discussion of the variations in color of the 
urine, it may be dark colored either on voiding or after standing. While 
such colorations may be due to melanin, they are much more frequently 
traceable to the presence of sulphuric acid in conjugation with phenol,^ 
paracresol, pyrocatechin, and hydroquinone. These substances are excreted 
especially in conditions associated either with increased intestinal putrefaction 
or with putrefactive processes elsewhere in the system. Aside from direct 
poisoning with these substances, they may be regarded as having much the 
same clinical significance as indican. 

QuaHtative and quantitative tests for these substances are rarely of clin- 
ical importance. They may be roughly determined by estimating the 
amount of ethereal sulphates in the urine, when a large increase may be as- 
sumed to be referable to these bodies unless indican is greatly in excess. 

(e) Alkapton. 

In certain conditions of disturbed protein metabolism, the urine contains 
pigments which cause it to turn dark on the addition of alkah or on standing. 
These urines are normal in color when voided and become black almost im- 
mediately on the addition of alkali, whence the name alkapton bodies and 

1 Biochem. Ztschr., 1910, XXVIII, 181. 

2 Urriola (Interstate Med. Jour., 191 2, XIX, 74) claims that a urinary excretion of blood 
pigment (probably hematin and not melanin) is invariably present as a pathognomonic sign 
of malaria. This may appear in the centrifuged specimen as a very abundant intense black 
pigment; a blue pigment, very constant but small in amount; or as an ochre pigment 
occasionally. 

2 See Hensel, Ztschr. f. physiol. Chem., 191 2, LXXVIII, 373; also, Folin and Denis, 
Jour. Biol. Chem., 1915, XXII, 305 and 309; Siegfried and Zimmermann, Biochem. Ztschr. 
1915, LXX, 124; Dubin, Jour. Biol. Chem., 1916, XXVI, 69; Anderson, Ibid., 387, 401 and 
409; Moore, Am. Jour. Dis. Child., 1917, XIII, 15; Dubin, Jour. Biol. Chem., 1917, XXXI, 
255; Tsudji, Ibid., 1919, XXXVIII, 13. For studies of phenols in blood see Benedict 
and Theis, Jour. Biol. Chem., 1918, XXXVI, 95 and 99. 



THE URINE 351 

alkaptonuria for their excretion in the urine. The substances causing this 
change of color are hydroquinon-acetic acid (as it has been prepared syn- 
thetically from gentisic aldehyd, it has been called homogentisic acid with the 
formula C6H3(OH)2(CH2COOH) and uroleucic acid, whose structure has not 
been absolutely settled.^ The former of these acids is present in all cases of 
alkaptonuria, while the latter may be present in all cases, but in such small 
amounts that it remains undetected. Urines containing these bodies strongly 
reduce copper and ammoniacal silver solutions, while bismuth solutions are 
little affected. The so-called glycosuric acid of Marshall is probably identical 
with homogentisic acid and is not a definite chemical entity. 

This . condition is observed at various periods of life and seems to be of 
theoretical more than practical interest, as of the 78 cases reported none ap- 
peared to be much disturbed in health, the condition usually being detected 
accidentally.^ 

The source of these substances is still uncertain. Baumann and Wolkow 
believe that the influence of specific bacteria in the intestinal canal upon the 
tyrosin, formed in the hydrolytic cleavage of the proteins, leads to direct 
formation of homogentisic acid. While this -accounts for some of the abnor- 
mal substance, it cannot account for the entire amount of 3 to 7 grams of 
homogentisic acid excreted in the 24 hours. From the work of Meyer, Falta, 
Langstein and Wohlgemuth it would appear that this condition depends not 
on an abnormal formation of homogentisic acid but on an incapacity for 
further oxidizing it when formed. Dakin^ believes that both factors play a 
part. However this may be, alkaptonuria has very few ill-effects, although 
transitory symptoms have been observed in occasional cases of diabetes, 
cirrhosis of the liver, tuberculosis, pyonephrosis and gastritis. The browning 
of the cartilage occurring in ochronosis is supposed by Albrecht and 
Zdarek to have some relation to the alkapton bodies, although Langstein 
could not show the alkapton acids in the urine in such cases. 

Qualitative and quantitative tests for these acids are rarely necessary. 
The characteristic change of color on alkalinizing the urine is, at least, sugges- 
tive of homogentisic acid. Like all other hydroxy derivatives of benzol, this 
substance shows the Schiff reaction with ferric chlorid, producing a transitory 
blue coloration when present in amounts of i to 4,000. The reduction of 
copper solutions should not be mistaken for sugar, as homogentisic acid 
neither ferments nor shows any optical activity. The urine will usually show 
a relatively high acidity. As this substance is practically harmless in the 
system we should expect to find, as we actually do, no increase in the conju- 
gated sulphates or glycuronates nor any increase in the ammonia content of 
the urine (Neuberg). 

^ See Morner, Ztschr. f. physiol. Chem., 191 2, LXXVIII, 306; Oswald (Ztschr. f. physiol. 
Chem., 1914, XCIII, 307) believes this acid is identical with homogentisic acid. See, also, 
Gross, Biochem. Ztschr., 1914, LXI, 165. 

2 See Poulsen, Munch, med. Wchnschr., 191 2, LIX, 364; also, Baldwin, Am. Jour. Med. 
Sc, 1913, CXLV, 123. 

'Jour. Biol. Chem., 1911, IX, 151. See, also, Pincussohn, Ergeb. d. inn. Med., u. d. 
Kinder heilkde., 191 2, VII, 454; Soderbergh, Nord. Med. Arch., 1915, XL VIII, i; 
Scheltema, Nederl. Tijdschr. v. Geneesk., 1915, II, 2659; Maggiore, Pediatria, 1916, 
XXIV, 234; Katsch, Deutsche Arch. f. klin. med., 1918, CXXVII, 210; Schochet, Arch. 
Int. Med., 1918, XXII, 82. 



352 DIAGNOSTIC METHODS 

(/) Ehrlich's Diazo Reaction. 

Under certain pathologic conditions the urine has been found to contain 
a chromogen which gives a deep red color to the urine when treated with 
diazo compounds and ammonia. This is known as the diazo reaction, the 
urinary substance causing it being somewhat uncertain. According to Bond- 
zynski, the alloxyproteic acid is the causative factor, but this needs confirma- 
tion, as Clemens has apparently shown that the body producing the reaction 
is sulphur-free. 

In the performance of this test two reagents are used: (i) sulphanilic acid 
5 grams, 50 ex. of concentrated hydrochloric acid, and 1,000 c.c. of water; 
(2) 3^ per cent, aqueous sodium nitrite solution. 

To a mixture consisting of 50 parts of solution i and one part of solution 
2 is added an equal volume of the urine. The mixture is then shaken and 
about one- tenth of the bulk of ammonia added quickly, and the mixture 
thoroughly shaken. The ammonia may be added in such a way that a line of 
contact forms, this latter method frequently bringing out a much more 
beautiful reaction. According to Greene, a mixture consisting of 100 parts of 
solution I to one of solution 2 renders the test more delicate. Instead of sul- 
phanilicacid,paraamidoacetophenonmaybeused,assuggestedbyFriedenwald. 

If the test be positive the entire urine will assume an intense red coloration 
or a colored ring will be observed at the point of contact between the ammonia 
and the mixture. With normal urine a distinct orange color may be observed. 
On shaking the mixture the foam will be more or less brilliant red in color, 
which is more characteristic than the red coloration of the mixture. On al- 
lowing the mixture to stand for 24 hours a green precipitate may be observed 
at the bottom of the tube, which Ehrlich regards as especially characteristic 
of the true diazo reaction. This green precipitate is not always present and is 
not necessary for a positive reaction, the red coloration of the mixture and 
of the foam being more frequent. 

The administration of certain drugs may markedly affect this test.^ Thus 
we find that naphthalin, chrysarobin, opium, and phenol derivatives give 
a reaction which is very similar to the true diazo reaction, but may be distin- 
guished, according to Wood, by the fact that color is more permanent in alka- 
Une solutions, does not fade to any extent on the addition of a strong mineral 
acid, the foam is more yellow than in the typhoid reaction, and the green pre- 
cipitate does not appear on standing. Burghart has found that, following 
the administration of tannic acid, gallic acid, tannigen, and tannalbin, the 
diazo reaction disappears from the urine, the inhibiting effect being perhaps 
exerted upon the reagents used rather than upon the unknown factor which 
usually causes the coloration. 

According to EhrHch, if the urine contains an excess of biliary pigment, a 
dark cloudy discoloration may occur, which is changed on boiHng to a distinct 
reddish violet. He also finds on applying the diazo test in some cases that the 
urine and foam become yellow before the addition of ammonia. After 

1 Skorczewski und Sohn (Wien. klin. Wchnschr., 191 1, XXIV, 1700) show that the urine 
of_'patients taking atophan gives this reaction. 



THE URINE 353 

ammonia is added the color changes to a Ughter yellow. This reaction is 
known as Ehrlich's "egg-yellow'' reaction, and is supposed to be due to the 
presence of urobilinogen. He regards this latter test as especially important 
in predicting the crisis of pneumonia. 

This diazo reaction is never positive in health. It was formerly regarded 
as pathognomonic of tj'phoid fever, but it has been show^n to occur in many 
other conditions. It is true that in typhoid fever it may be present as early 
as the third or fourth day and may persist for some time, reappearing if a 
relapse occurs, while the Widal test, as will be remembered, does not vary 
with a relapse. Moreover, the intensity of this reaction is somewhat parallel 
with the severity of the case, while the Widal reaction may not be present 
in the severest types of t^^phoid fever. A positive diazo reaction occurs fre- 
quently in measles, somew^hat less frequently in pneumonia, miliary tubercu- 
losis, scarlet fever, ^ diphtheria, and erysipelas, while in rheumatism and 
meningitis it is rarely obtained. Michaelis believes that the presence of a 
positive diazo reaction in pulmonary tuberculosis indicates a progressive 
condition with a grave prognosis.^ 

This reaction is, therefore, a valuable aid in diagnostic work. As it is 
present in about 80 per cent, of cases of typhoid fever, a negative reaction 
would not necessarily exclude typhoid nor would a positive reaction prove 
its presence. Its appearance in a relapse is of value, as the Widal test would 
give, no information under such conditions. It is to be said, however, that 
the diagnosis should by no means rest upon this test. 
ig) Russo'sTest. 

This test^ has been recently advanced and seems to have somew^hat more 
diagnostic importance than has the diazo reaction. The technic is very 
simple and is as follows: Four drops of a i to 1,000 aqueous solution of meth- 
ylene blue are added to 4 or 5 c.c. of suspected urine. If the reaction be posi- 
tive the mixture turns to an emerald or mint-green hue. A light-green 
or bluish-green tint shows a negative reaction. The positive reaction is not 
affected by boiling the urine or by the previous ingestion of such compounds 
as phenacetin, salol, quinin, and calomel. The difficulty in the application 
of the test comes in the ability to recognize the various tints of green w^hich 
may be present. With a little practice, however, a positive reaction may be 
readily detected, especially if a control test be made with normal urine. 

This test is shown as early as the second day of typhoid fever and persists 
throughout its course. The mint-green hue is first observed, the emerald- 
green tint appearing as the disease progresses. If the course is favorable 
the color tone becomes more and more bluish, while if unfavorable the 
emerald tint persists. This test is also given in measles, smallpox, chronic 

1 See Umber, Med. Klin., 1912, VIII, 322; Woody and Kolmer, Arch. Pediat., 1913 
XXIX, 18; Marcantoni, Gazz. d. osp., 1914, XXXV, 193; Jahrb. f. Kinderh., i9i5,LXXXI, 
168. 

- See Heflebower, Am. Jour. Med. Sc, 191 2, CXLIII, 221; Pick, Med. klin., 191 5, XI, 
1292; Levy, Deutsche med. Wchnschr., 1915, XLI, 1212; Bosch, Ibid., 1916, XLII, 17; 
Sinclair, Jour. A. M. A.. 1916, LX\T, 247; Corper and Callahan, Jour. Lab. and Clin. Med., 
1916, I, 740; Pottenger, Ibid., II, 37. 

^Riforma Medica, 1905, XXI, 507; abstract Jour. x\m. Med. Assn.. 1905, XLV, 363; 
Kahn and Wechsler, Med. Record, 1916, LXXXIX, 106. 



354 



DIAGNOSTIC METHODS 



and suppurative tuberculosis, but is negative in varioloid, varicella, scarlet 

fever, miliary tuberculosis, appendicitis, and malaria. 

This test is as simple and just as reliable as is the diazo-reaction, being 

especially valuable in differentiating a typhoid from a mihary tuberculosis. 

Rolph and Nelson^ have pointed out that urines containing bilirubin react to 

this test, so that this fallacy must be borne in mind. 
{h) Dimethylaminobenzaldehyd Reaction. 

This test, advanced by Ehrlich, is as follows: Prepare a 2 per cent, solution 

of p-dimethylaminobenzaldehyd in 
equal parts of concentrated HCl and 
water. Add a few drops of this solution 
to 5 c.c. of fresh cold urine and allow to 
stand for a few minutes. A positive 
reaction is indicated by the appearance 
of a cherry-red color, which may be 
extracted with chloroform or epichlor- 
hydrin. Heating facihtates the re- 
action, but here normal urine may give 
a slight reddish coloration. In the cold 
normal urine gives a greenish-yellow 
color. Apparently this coloration is 
due to the presence of metabolic prod- 
ucts derived from blood pigments 
(urobilinogen compounds, see p. 269). ^ 
It is to be expected, therefore, that this 
reaction would be distinct in diseases of 
the liver and bile passages, although it 
is not constant even here. It is, also, 
observed in tuberculosis, pneumonia, 

typhoid fever, malaria and scarlet fever. ^ It has little clinical value. 




Fig. 



-Purdy electric centrifuge. 



IV. Microscopic Examination of the Urine. 
The microscopic examination of the urine is important in every case. 
So varied are the elements which appear in the microscopic field that con- 
siderable experience is necessary before absolute interpretation can be made. 
Not infrequently the character of the sediment wiU change a diagnosis, as, 

^ Medical Record, 191 1, LXXX, 373. See, also, Neuman and Behrend, Arch, Int. Med., 

1913, XI, 456; da Pozzo, Gazz. d. osp., 1914, XXXV, 865; Petzetakis (Lyon Med., 1916, 
CXXV, 309) reports a similar test performed by the addition of two or three drops of a 5 
per cent alcoholic solution of iodin to 10 to 15 c.c. of urine. A golden yellow tint is a posi- 
tive reaction, which he claims is shown in "open" tuberculosis and typhoid fever, but not in 
"closed" tuberculosis. See, Ruiz and Moliner, Arch. Esp. de Pediat., 1919, III, 420. 

2 Fischer and Meyer-Betz (Ztschr. f. physiol. Chem., 191 1, LXXV, 232) show that the 
reaction depends on the presence of a dipyrrylphenylmethane pigment of the class of non- 
stable pyrrol derivatives, to which group the blood and biliary pigments, as well as urobilino- 
gen, belong. 

3 See Hesse, Med. Klin., 1913, IX, 294; also, Steensma, Nederl. Tijdschr. v. Geneesk., 

1914, LVIII, 467; Rabinowitsch, Berl. klin. Wchnschr., 1914, LI, 1456; Berkowitz, Med. 
Record, 1914, LXXXVI, 1087; Eisner, Deutsch. Ztschr. f. Chir., 1915, CXXXII, 589; 
Robertson, Calif. State Jour. Med., 191 5, XIII, 65; Litzenberg (Chicago Gynec. Soc, 
Apr. 16, 1 91 5) reports an increase of urobilinogen in the toxemias of pregnancy. 



THE URINE 



355 



for instance, when pus- or blood-cells are present in sufficiently large amounts 
to account for an albuminuria previously determined by chemical methods. 
It is, therefore, essential that the microscopic examination of urine form a 
part of the ordinary routine. 

In obtaining the sediment of the urine for microscopic examination, two 
methods are possible. In the first place, the urine may be allowed to stand 
in a conical glass for periods ranging from 12 to 24 hours. The sediment 
originally present in the urine as well as that formed by chemical changes 
taking place during the standing will collect in the lowest portion of the 
glass and may be removed by a glass-tube drawn out to a somewhat small 
point. It is to be remembered that a sediment at the end of 12 to 24 hours 
may be entirely different from that originally present in the freshly voided 
specimen. The changes in the reaction of the urine will, necessarily, lead to 
the dissolving of certain types of crystals and to the formation of other 
varieties. Moreover, casts, if originally present, may dissolve or disappear 
as a result of the reaction of the urine becoming alkaline. For these reasons 
it is absolutely essential, in the use of this gravity method, that preservatives 
be added to the urine. Among the preservatives which may be used to pre- 
vent bacterial action during the sedimentation, we find a small piece of cam- 
phor or a rather large crystal of thymol serving the purpose. Some workers 
add one-fifth the volume of a 4 per cent, solution of borax which is equally 
useful, but the addition of chloroform or for- 
malin, does not serve as well in these cases, 
as the former does not completely preserve the 
casts and the latter introduces a crystalline 
compound of formalin and urea which may be 
confusing, as it is not unlike impure leucin. 
This method is not to be recommended for 
routine work, as it requires too long a period 
and as changes may occur which make it ab- 
solutely impossible to differentiate between a 
primary and a secondary sediment. 

Secondly, the sediment may be thrown 
down by the use of the centrifuge. This ap- 
paratus is seen in the accompanying cut. By 
the use of this method no preservative is 
needed, a deposit is obtained within three min- 
utes in a much more concentrated form, and 
changes in the sediment do not take place. 
The writer would recommend, therefore, the 
use preferably of the electric centrifuge or, at 
least, of the type run by hand. 

Whatever method may be adopted for 
obtaining the urinary sediment, the next steps in the process are the same. 
A pipet, consisting of a glass tube drawn out to a point about one-half the 
diameter of the tube, is introduced to the bottom of the vessel containing the 



Fig. 90. — 
Sediment tube 
for Purdy cen- 
trifuge. 



Fig. 91. — Per- 
centage centri- 
fuge tube. 



356 DIAGNOSTIC METHODS 

sediment, a finger being placed over the upper end to prevent fluid passing 
into the tube as it is introduced. When the tip of the pipet comes in contact 
with the deposit, the pressure of the finger on the upper end of the pipet is 
removed and the deposit allowed to flow up into the pipet. The finger is then 
placed tightly over the tube as it is withdrawn from the fluid. By placing 
the tip of the pipet in contact with a perfectly clean slide which is absolutely 
free from scratches and by gradually rotating the pipet, a small portion of the 
sediment is obtained. A cover-glass is then placed upon this drop avoiding 
any undue pressure, which might distort the organized elements of the 
sediment. Some workers dispense with the cover-glass and use somewhat 
larger amounts of sediment, but the writer does not find this method as 
acceptable owing to the fact that the lens of the objective may dip into the 
fluid and thus give indefinite microscopic pictures. Moreover, the focus 
cannot be as accurately adjusted without the use of the cover-glass. 

In the examination of the microscopic specimen, prepared as above, 
the point of special importance to be observed is the proper adjustment of the 
light. The writer is accustomed to use the low-power objective in the pre- 
Uminary examination. In this case it is essential that the light be shut off 
to a large extent, as the recognition of casts cannot be made in a brilliantly 
illuminated field. This examination with the low power has the advantages 
that larger visual fields are subject to inspection, casts are easil} recognized 
and crystalline deposits, as well as morphological elements, are usually differ- 
entiated. After the preliminary examination with the low power, the final 
examination is made with the high-power dry lens. In this way elements 
which appear suspicious under the low power are more clearly brought out 
and differenjtiations made possible between various types of cellular elements. 
With the high-power lens it is, of course, essential that the field be somewhat 
more illuminated than when the low-power is used. While a mechanical 
stage is, at times, advantageous in the microscopic examination of the urine, 
the writer has found that the fingers serve practically every purpose in the 
manipulation of the slide under examination. 

Where the urine is to be examined for the presence of bacteria, stained 
specimens must be made and examined with the oil-immersion lens. 

Urinary sediments are classified into two divisions: (a) chemical or 
nonorganized and (b) anatomical or organized sediments. The nonorganized 
sediments exist in solution in normal urine and appear as deposits under 
conditions of excessive formation, excessive excretion, or of alterations in the 
urine affecting its solvent properties. The chief chemical sediments are 
uric acid and its salts, calcium oxalate, phosphates, sulphates, cystin, leucin, 
tyrosin, xanthin, fat, and fatty acids. The organized sediments are usually 
foreign substances and are not met with in normal urine. They consist of 
epithelial cells, pus corpuscles, blood-cells, renal casts, spermatozoa, infusoria, 
bacteria, and tissue fragments. 
{A) Unorganized Sedknents. 
(a) Those Appearing in Acid IJrine. 
(i) Uric Acid (C5H4N4O3). 

This occurs as a sediment in the urine under 3 conditions: (i) great 



THE URINE 



357 



concentration, (2) high acidity, and (3) low temperature. The deposit differs 
from others in possessing a deep yellow or orange-red color, although some 
of the smaller crystals are occasionally colorless. The primary form of the 
uric acid crystal is that of the rhombic prism. Modifications of this, in the 
form of square plates, cubes, ovoids, dumb-bells, or whetstone crystals are 
sometimes noticed. A rare type, especially of the colorless crystals, is a per- 
fect hexagon which resembles cystin so closely that chemical means of differ- 
entiation must be used. The crystals may be single or grouped in rosettes or 
fan-shaped masses. Occasionally typical needle-shaped crystals may be seen 
which are arranged in sheaves. " 

The microscopic peculiarities of uric acid are usually such as to permit 
of its easy recognition. In some cases, however, it is wise to confirm the 




Fig. 92. — Various forms of uric acid, i, Rhombic plates; 2, whetstone forms; 3, quad- 
rate forms; 4, 5, prolonged into points; 6, 8, rosettes; 7, pointed bundles; 9, barrel forms 
precipitated by adding hydrochloric acid to urine. (Hawk.) 

microscopic finding by the murexid test as follows : Place a small quantity of 
the sediment in an evaporating dish and add a few drops of concentrated 
nitric acid. Evaporate on the water-bath to dryness, when a yellowish or 
reddish residue will remain. Allow the residue to cool and add a few drops of 
ammonium hydrate solution. In the presence of uric acid a distinct reddish 
purple color will appear. If water be added to this purple solution and the 
mixture evaporated to dryness the color disappears. This latter point is 
of importance as xanthin, which may resemble unusual types of uric' acid in 
microscopic appearance, also gives the murexid test, but the color does not 
disappear on heating with water. ^ 

(2) Sodium Acid Urate (C5H3NaN403). 

This salt of uric acid forms the bulk of the "brick-dust deposit" or ''sedi- 
mentum lateritium" found when urine has cooled. In such cases the urine 
\See Kohler, Ztschr. f. klin. Med., 1919, LXXXVII, 338. 



358 



DIAGNOSTIC METHODS 



first shows a milky appearance and the sediment soon settles on the sides 
and bottom of the container. This deposit is usually in the form of irregular 




Fig. 93. — Acid sodium urate. {Hawk.) 

amorphous granules of a brownish or pink color. Occasionally the sediment 
may be distinctly crystalline, occurring as prismatic needle-like crystals 
which are grouped in star-shaped, fan-shaped or dumb-bell-like clusters. 




Fig. 94. — Xanthin. {Hawk.) 

(3) Potassium Acid Urate (C5H3KN4O3). 

This substance occurs only as a granular amorphous deposit. Owing 
to its greater solubility, it does not form as large an amount of the brick-dust 
deposit as does the sodium salt. 

These two latter sediments are occasionally associated with amorphous 
deposits of the calcium and magnesium acid urates. These are, however, 
rare and need not be separately considered. In detecting the presence of the 
urates in a deposit, a small portion of the turbid urine is poured into a 
test-tube and gently heated. If urates are present the sediment will com- 
pletely dissolve. These salts also give the murexid test. 



THE URINE 359 

(4) Xanthin (C5H4N4O2). 

This substance which is chemically closely related to uric acid is rarely 
found as a sediment in the urine. Its chief clinical importance is found in 
its appearance as a urinary calculus. It crystallizes in whetstone-shaped 
colorless crystals which resemble those of uric acid, from which it is differen- 
tiated by its solubility on heating and in hydrochloric acid as well as ammonia. 
It may be chemically recognized by WeideVs reaction. Place a portion of 
the suspected crystalline deposit in an evaporating dish and dissolve by warm- 
ing with a few drops of bromine water. Evaporate to dryness and place the 
dish containing the residue under a large beaker, allow the fumes of ammonia 
to fiU the inverted beaker when a red or purpUsh- violet color will be produced 
in the presence of xanthin. 

(5) Calcium Oxalate (CaC20 4). 

This substance appears most frequently in acid urine, but may be found 

.® o f - ^ , 

Fig. 95. — Calcium oxalate. (Hawk after Ogden.) 

after the urine has undergone alkahne fermentation. If it occurs in acid urine 
it is associated with uric acid; if in alkaline urine, with the triple phosphates. 
The deposit is a colorless crystalHne one having two distinct forms: (i) octahe- 
dral crystals (four-sided pyramids lying base to base) ; viewed from the side, 
these appear as squares crossed by two sharp lines, giving the so-called "en- 
velope" crystal. (2) Dumb-bell crystals in the form of ovoid or circular 
disks with round margins depressed at the centers. These latter often pre- 
sent radial striations. Emerson has called attention to a rare type of cal- 
cium oxalate crystal which appears in the form of flat plates with parallel 
sides and rounded ends, looking Uke superimposed sheets of mica. It is 
characteristic of the crystallization of this substance, as of most crystalline 
urinary deposits, that the crystals are practically always of the same type, 
variations rarely appearing in the same specimen of urine. 

These crystals are insoluble in acetic acid, but soluble in hydrochloric 
acid. This is a point of some importance, as it is occasionally difficult to 
distinguish microscopically between calcium oxalate and some crystals of 
triple phosphate. This latter crystal is soluble in acetic acid. These crystals 
may be chemically identified by dissolving them in hydrochloric acid, alkalin- 
izing with ammonium hydrate, and precipitating with ammonium oxalate. 



360 DIAGNOSTIC METHODS 

(6) Cystin (C3H6NS02)2. 

The appearance of cystin in the urine is known as cystinuria. This is 
a condition of perverted protein metabolism which is not well understood. 
It may make its appearance at any period of life and in either sex, perhaps 
somewhat more frequently in the male. It shows a remarkably frequent 
hereditary character, being observed in some cases through several genera- 
tions and in several children of the same parents. Many of these cases do not 
show any chnical characteristics, being present during their entire life without 
any apparent symptoms. In other cases, owing to the formation of calculi, 
frequent manifestations are noted and surgical intervention interposed. 
This condition is not very frequent, being reported only 180 times in the 
literature.^ 

"Prior to the time when cystin was found to be a product of the disinte- 
gration of protein substances it had been conjectured, on the ground of its 
content in sulphur and nitrogen,that it might be a product of the intermediate 
protein metabolism. The explanation which von Udranzki and Baumann 
gave of its excretion corresponds in principle to that which is familiar to-day 



/ 





Fig. 96. — Cystin. {Hawk after Ogden.) - 

for the appearance of conjugation products of glycuronic acid and of glycocoll 
in urine. Like the latter, it should be normally further oxidized, and only 
in the presence of definite bodies should it be intercepted. These authors 
considered the binding substances to be the previously mentioned diamins, 
especially putrescin and cadaverin, which they had found in the urine and 
in the feces of a cystinuric patient. The formation of the diamins was sup- 
posed to be brought about by specific bacteria in the digestive tract by an ex- 
traordinary chronic intestinal mycosis. The resorbed part of the cadaverin 
and putrescin was supposed to protect the cystin from combustion, just as 
benzoic acid does glycocoll, by entering into a loose combination which de- 
composes again after passing through the kidneys. Serious difficulties, how- 
ever, have opposed themselves to this interpretation of cystinuria. First, 
numerous cases have been described without simultaneous diaminuria, and, 
conversely, diaminuria occurs in malaria, and in the conditions brought about 
by the cholera vibrios and the Finckler-Prior bacillus, without cystinuria 
having ever set in, any more than it does when diamins themselves are 
administered." 

^ See Fromherz, Bed. klin. Wchnschr., 1913, L, 1618; Neumann, Deutsch. med. Wchn- 
schr., 1914, XL, 2065. 



THE URINE 



361 



"Further investigations have shown that cystinuria is really a disturbance 
of the amino-acid metabolism. Of the end-products of protein hydrolysis 
which arise in the system, the cystinuric cannot avail himself in the normal 
way of the cystin, and in part excretes it; the remaining products of protein 
hydrolysis undergo their ordinary fate. If free monomolecular «-amino acids 
appear in places which are at present not well known, or if they occur there 
even in unusual amounts, then, unlike the normal individual, the cystinuric 
is unable to burn them and they leave the organism unchanged just as cystin 
itself does. The basic diamino acids behave in practically the same way, 
except that the CO2 group is split off from them and we arrive at diaminuria" 
(Neuberg). 

Crystals of cystin are rare in the urinary sediment. In some of the cases 
reported the cystin did not separate from the urine until this was acidified 
with acetic acid and allowed to stand for 12 hours. It crystallizes in two 




Fig. 97. — Pure leucin. (Hawk.) 

forms: (i) six-sided tablets having an opalescent luster and sometimes 
traced with fine fines of secondary crystaUization; (2) four-sided square 
prisms lying separately or in steUate forms. These crystals are soluble in 
hydrochloric acid, alkafine hydrates, and insoluble in acetic acid. These 
tests differentiate it from uric acid. If the urinary sediment suspected of 
containing cystin be treated with strong sodium hydrate solution and a few 
drops of benzoyl chlorid, and the mixture shaken, a voluminous precipitate 
of benzoyl cystin is obtained. 

(7) Leucin (CeHisNOs). 

Chemically leucin is a-aminoisobutylacetic acid. It occurs in the urine 
in conditions associated with more or less marked derangement of hepatic 
functions (see amino acids). As found in the urine, leucin appears in the 
form of yellowish, highly refractile spherules, with alternating light and dark 
concentric layers and with radial striations. . In the pure state it crystallizes 
either in thin, white, hexagonal plates or as scales or rosettes of irregular 
shapes. 



362 



DIAGNOSTIC METHODS 



Leucin is soluble in water, acids, and alkalies, and insoluble, to a more or 
less extent, in alcohol. Not always do we find crystals of leucin in the sedi- 
ment when the urine contains this substance. If it be suspected, the urine 
should be evaporated to a small bulk and alcohol added to the residue, which 
may then be examined for the characteristic crystals. This leucin may be 
identified by Scherer^s test as follows : Some of the solid residue obtained by 
concentrating the urine to a small bulk is evaporated with concentrated nitric 
acid on a platinum crucible cover. With pure leucin the residue remains 




Fig. 98, — Impure leucin. (Hawk after Ogden.) 

colorless, but as usually applied to the urine a yellowish residue obtains. 
This is heated with a few drops of sodium hydrate solution, when a yellowish 
or brown color will be observed. If further heating be applied the leucin will 
collect into an oily drop which rolls around on the heated surface. As leucin 
does not stain with Sudan-Ill it should not be confused with fat. 

(8) Tyrosin (C9H11NO3). 

Chemically, tyrosin is p-oxyphenyl-a-amino-propionic acid. As found 




Fig. 99. — Tyrosin. {Hawk.) 

in the urine tyrosin crystallizes in the form of fine colorless needles, which 
may appear black and are arranged in sheaf-hke collections or rosettes. Like 
leucin, it may not crystallize out unless the urine be concentrated. Tyrosin 



THE URINE 363 

is soluble in water, acids, and alkalies, while it is slightly soluble in alcohol 
and insoluble in ether. As other crystals, which may appear in the urine 
closely resemble the tyrosin needles, it is advisable to confirm the microscopic 
findings by chemical tests. ^ This may be done by evaporating the urine to a 
small bulk, removing the fluid and dissolving the residue in water. Morner^s 
test may then be applied as follows: To this aqueous solution is added i c.c. 
of a reagent consisting of i c.c. of formalin, 55 c.c. of concentrated sulphuric 
acid, and 45 c.c. of water. If the mixture be heated to boiling a beautiful 
green color will be observed in the presence of tyrosin. 

(9) Calcium Sulphate (CaS042H20). 

This is a very rare sediment, appearing only when the urine is extremely 
acid. The crystals appear in the form of long, thin, rhombic plates or needles 
which may be single, but are more frequently observed in clusters. If the 
sediment be boiled with hydrochloric acid and barium chlorid added, a pre- 
cipitate of barium sulphate will point to the presence of calcium sulphate in 
the sediment. 




« 



Fig. 100. — Calcium sulphate. (Hawk Fig. ioi. — Bilirubin (Haematoidin) . 

after Hensel and Weil.) (Hawk after Ogden.) 

(10) Bilirubin (CieHigNsOs). 

Bilirubin or its isomer hematoidin may appear in the urine in conditions 
previously discussed. The type of crystal is either a brilliant yellow or ruby- 
red rhomb or a yellow needle. Rarely the deposit may be in the form of a 
yellow granular sediment. Not infrequently small curved needle-like spines 
are observed projecting from the angles of the rhombic crystals. These 
crystals may be identified by extracting the acid urine with chloroform and 
applying the tests previously discussed under BiUary Pigments. 

(11) Hippuric Acid (C9H9NO3). 

This substance has been observed as a sediment, although rarely. It ap- 
pears in the form of semitransparent, colorless, four-sided prisms, or in long 
pointed rods or needles, occasionally in forms closely resembling those of the 
triple phosphates to be described later. These crystals are soluble in warm 
water, alcohol and ether and may be distinguished from uric acid by the fact 
that they do not give the murexid test. 

^ See Rosenbloom and Gardner, New York Med. Jour., 1 914, C, 574. 



364 • DIAGNOSTIC METHODS 

(12) Neutral Calciiim Phosphate (CaHP042H20). 

This substance is found only in faintly acid or neutral urine. It is quite 
rare as a sediment, crystallizing in colorless needles or slender pyramids which 
group themselves together with their points in a common center to form 
rosettes or cross-shaped figures. These crystals are soluble in acetic acid and 
may be converted into calcium carbonate when treated with a strong solution 
of ammonium carbonate. 

(13) Fat. 

Under normal conditions the urine contains no free fat, but amounts 
varying from traces to rather large excretions may be found under pathologic 
conditions.^ The excretion of fat in the urine is known as lipuria. This is 
characterized by the presence of small or large strongly refractile globules 
which may be stained black with osmic acid or red with Sudan-Ill. These 
globules are soluble in ether and may, therefore, be extracted from the urine 
by shaking out with this solvent. It not infrequently happens that the urine 
is contaminated with fat which may have been used in obtaining catheterized 
specimens or with fat coming from the bottle containing the urine. This 
may lead to a diagnosis of lipuria unless care be taken to exclude such a 
source. True lipuria has been observed in various conditions. Thus we may 
find after a large intake of fat in the diet, or as a therapeutic agent the so-called 
^^ alimentary lipuria.''^ Pathologically, it has been observed in various 
cachectic conditions, in crushing injuries, especially of the bones, in eclampsia, 
in chronic heart disease, fatty tumors, diabetes mellitus, tuberculosis, various 
affections of the pancreas and liver, nephritis, and after the use of various 
general protoplasmic poisons. In these cases the blood may also contain an 
excess of fat, although this has not been observed in aU cases. In fatty 
degeneration along the genitourinary tract, fat droplets may be seen in the 
epithelial cells and in the casts. Free fat is rarely found in such conditions, 
but occasionally it may collect in droplets which float on the surface of the 
urine and then constitutes true lipuria. Occasionally flat superimposed 
plates with notched corners (cholesterin) may be seen and may be so numer- 
ous as to justify the term "cholesterinuria." 

In conditions associated with infection by the filaria, large amounts of fat 
may be present, giving rise to the appearance of an emulsion. To this con- 
dition has been given the name chyluria. In this form the fat may be present 
occasionally in large masses resembling tallow, but more frequently is seen in 
finer clumps of globules. The appearance of the urine is much like that of 
skimmed milk, but may have a reddish tinge due to the presence of blood. 
On allowing the urine to stand, a cream-like mass of fat will rise to the sur- 
face. It is not unusual in such cases to find the ova or the parasite in the 
masses of coagulated material. The excretion of the fatty material at times 
runs a somewhat cyclic course, being present during the day and absent at 
night or vice versa. Occasionally the excretion varies with the position of the 
patient, being somewhat more frequent when he is erect, and may be mark- 

1 See Sakaguchi, Biochem. Ztschr., 1913, XLVIII, i ; Lawrynowicz, Ztschr. f. klin. Med., 
1914, LXXX, 389. 



PLATE X. 




Ammonium Urate, showing Spherules and Thorn-apple-shaped Crystals. 
(From Ogden, after Peyer.) 



THE URINE 



365 



edly increased after severe exercise. This condition should be taken simply 
as a symptom of filariasis. 

A nonparasitic type of chyluria has been observed, but its etiology is 
somewhat uncertain. It probably is closely related to the conditions above 
mentioned as causing true lipuria. It does have some relation to an increased 
fat diet and apparently is associated with exudation from the lymphatic 
vessels, as the cellular elements are largely lymphocytes.^ 




Fig. 102. — Cholesterin. {Hawk.) 

(b) Those Occurring in Alkaline Urine. 

(i) Ammonium Urate (C5H3(NH4)N403). 

This sediment occurs most frequently in combination with amorphous 
calcium phosphate and triple phosphate crystals. It is the only urate deposit 
found in alkaline urine, but may occur in neutral urine. It appears as a 
crystalline deposit of dark brown spherical masses studded with fine spiculae, 
from which fact the name of "thorn-apple'' crystals has been given to them. 
Occasionally these spheres may show concentric or radial striations. Not in- 
frequently one observes crystals having irregular shapes, such as those of a 
dumb-bell or a pear. 

Chemically, these crystals may be identified by dissolving in hydrochloric 
acid, when uric acid, which may be identified by the murexid test, will sepa- 
rate. If sodium hydrate be added to the dry sediment and heat applied, 
vapors of ammonia are given off. 

(2) Calcium Triphosphate (CasCPO 4)2). 

This compound is frequently found in alkaline urine, as a white amorphous 
flocculent deposit arranged in irregular patches. This is the usual deposit 
which appears in the urine when it becomes alkaline after meals. In the so- 
called ^' phosphaturia'' the urine is always turbid when voided so that the 
assumption was made that an excess of phosphoric acid was being excreted. 
Such is found not to be the case, as a deposition of the normal phosphates of 

^ See Stern, Arch. Diagnosis, 1913, VI, 114; Young, Jour. Trop. Med. and Hyg., 1914, 
XVII, 241; Sanes and Kahn, Arch. Int. Med., 1916, XVII, 181; Carter, Ibid., XVIII, 541; 
Hymanson, Am. Jour. Dis. Child., 1916, XI, 455. 



366 DIAGNOSTIC METHODS 

the urine must occur when the reaction becomes alkahne. It is to be said in 
this place that no conclusion whatever can be drawn from the separation of a 
substance in the sediment as regards an increase in its excretion. So many 
factors influence the separation or nonseparation of a sediment that a finding 
should not be regarded as evidence of increased formation and excretion 
unless quantitative chemical examination points in this direction. 

Calcium phosphate is soluble in acetic acid without evolution of gas, which 
test may be used to show the presence of this substance in the deposits. It 
may be absolutely identified as a calcium compound by dissolving in acetic 
acid and precipitating with ammonium oxalate; the phosphoric acid radical 
may be proven by dissolving the sediment in nitric acid and precipitating with 
ammonium molybdate. 

(3 ) Magnesium Phosphate (Mgs (PO 4) 2) . 

Theoretically this compound appears along with calcium phosphate as an 
amorphous deposit in alkaline urine. Its amount is, however, usually less 
than the latter compound. 

It is observed in rare cases as large, long, rhombic plates with beveled 
edges which closely resemble the crystals of triple phosphates. These 
crystals are found in cases in which not sufficient ammonia is present to form 
the true triple phosphate, and may be considered as transition crystals. 

(4) Magnesium-ammonium Phosphate (Mg(NH4)P04). 

The appearance of this substance in the urine is essentially characteristic 



^ 



\ 




Fig. 103. — Magnesium-ammonium phosphates. {Hawk after Ogden.) 

of ammoniacal urine. It may very rarely be seen in amphoteric urine when 
ammonium salts are present in large amounts. The crystals belong to the 
rhombic system, appearing most frequently as triangular prisms or "coffin- 
lid" crystals. These may be shortened in the form of squares or one or more 
corners may be rounded or beveled. By refracted light a greenish tone is 
observed when these crystals are present. A second type of the '' triple phos- 
phate" is that of a star-shaped feathery crystal with points not unlike fern 
leaves. These crystals are easily soluble in acetic acid and may be identified 
by treating with sodium hydrate and warming when ammonia is evolved. 



THE URINE 



367 



(5) Calcium Carbonate (CaCOs). 

This substance frequently occurs in alkaline urine in association with 
the amorphous phosphates. It may appear as groups of amorphous material 
or may form large spheroidal masses with concentric radiations. Occasionally 
it may be observed in dumb-bell like masses which resemble somewhat the 
same type of calcium oxalate crystal, from which it may be differentiated 
by the fact that it is soluble in acetic acid with production of CO2, while 
calcium oxalate remains undissolved. 




Fig. 104. — Calcium carbonate. {Hawk.) 

(B) Organized Sediments. 
(i) Mucoid material. 

Mucus is a constituent of practically every specimen of urine, in the 
form of the "nubecula." This appears in the form of small threads which 
branch and interlace in such a way that the entire microscopic field may be 
practically taken up by this material. In the meshes of the nubecular 
threads are observed the so-called "mucous corpuscles," which are prac- 
tically identical with the ordinary leucocyte. Little signifiance is attached 
to this form of mucous threads unless a great increase is observed, when it 
indicates, as does mucin, a vesicle catarrh. 

(2) Epithelial Cells. 

Normally, the only epithelial cells found in the urine are the irregular 
flat cells from the bladder and urethra or the large flat epithelia seen in the 
urine' of women and arising from the vagina. The presence of large num- 
bers of other types of epithelial cells is always pathological and denotes an 
inflammatory or destructive lesion somewhere along the genitourinary 
tract.^ It is a matter of more or less difficulty absolutely to identify, in all 
cases, the source of the epithelium found in the urine. According to Heitz- 
mann, the positive recognition is based largely upon the size of the cell, as the 
shape may vary from pathologic conditions as well as from the portion from 

1 See Schkarin, Russk. Vrach, 1915, XIV, 505. 



368 



DIAGNOSTIC METHODS 



which they are derived. As stratified epithehum is found in the pelvis of the 
kidney, the ureters, bladder, and urethra, it is to be expected that large flat 
cells, cuboidal cells, or columnar cells will appear depending upon the layer 
from which the cell is derived. As the simple epithelium exists in the urinif- 
erous tubules, the prostate gland, seminal vesicles, and ejaculatory ducts, the 
recognition of such cells will limit their origin, the size being important in de- 
termining the exact point from which they are derived. It is to be remem- 
bered, therefore, that the shape of the cell is of far less importance than is its size. 
The epithehal cells derived from the bladder are usually the large flat 
irregular cells commonly seen in all normal urine. They have a clear pro- 
toplasm and usually a small distinct central nucleus and are extremely gran- 
ular. These flat epithehal cells may be single, in groups, or if the irritation 
is marked may occur in large sheet-like masses. The large cuboidal cells of 




Fig. 105. — Epithelium from different areas of urinary tract, a, leucocyte (for com- 
parison); b, renal cells; c, superficial pelvic cells; d, deep pelvic cells; e, cells from calices; 
, cells from ureter; g, squamous epithelium from bladder; h, neck of bladder cells; i, epi- 
thelium from prostatic urethra; k, urethral cells; I, scaly epithelium; m, w^, cells from 
seminal passages; n, compound granule cells; 0, fatty renal cell. (Hawk after Ogden.) 

the bladder epithelium may be seen in acute cystitis in which they are asso- 
ciated with large numbers of the flat cells previously mentioned. If the 
conditions become chronic the flat cells may entirely disappear and be 
replaced by cuboidal and by a few columnar epithelial cells. These latter 
cells are especially observed in the severe inflammatory processes in the 
bladder. 

The large, flat, squamous epithehal cells derived from the vagina are 
more frequently arranged in stratified groups so that their recognition is 
usually simple. As these types of cells denote simple desquamation, being 
pathologic only when present in enormous numbers, an absolute differentia- 
tion is of little consequence clinically and, if it be so, the clinical symptoms of 
the case will usually clear up the decision. 

The urethral epithelium very closely resembles that above described. 



THE URINE 369 

The cells are large and irregular, being partly flat, partly cuboidal and partly 
columnar. The cylindrical types of urethral epithelium may occur in the 
form of longer, irregular, smaller types, than those of the bladder or vagina. 
This type constitutes the so-called "tailed" cells, which may be derived from 
the pelvis of the kidney and were at one time held to be indicative of a pyelitis. 
Sahli regards a preponderance of such tailed cells over the flatter and more 
regular types as distinct evidence of trouble in the renal pelves. As these 
cells may be derived from other portions of the urinary tracts it is unwise 
to make an absolute diagnosis on such a finding. The small polygonal cells 
as well as tailed cells may be derived also from the ureter so that our diagnosis 
would necessarily rest upon findings other than such epithelium. 

In the writer's opinion, it is a practical impossibility to make a positive 
diagnosis of a lesion in any specific portion of the genitourinary tract based 
entirely upon the appearance of the urinary epithelium. The points to be 
remembered are that we may have any type of epithelium and may have 
many variations in shape as well as in size. Such variations may be present 
in any portion of the urinary system, although distinctly renal epithelial cells 
are more frequently in the form of round or cubical cells somewhat larger 
than the leucocyte and containing a large vesicular nucleus. These latter 
renal cells are the only ones which seem to the writer distinctly diagnostic. 
They are differentiated from the similar cells arising from the ureters and 
prostate gland by the fact that the latter cells are about twice the size of the 
pus-cell, being consequently larger than the true renal cell. 

Degenerative_ changes are frequently observed in these epithelial cells, 
even when examined immediately after voiding. The usual type of this de- 
generation is the presence of fat granules or globules, especially in the small 
renal cells. If the sediment be treated with Sudan-Ill these granules will 
appear distinctly red. 

(3) Pus-cells. 

A few leucocytes may be observed in practically every specimen of urine, 

especially in those from women, in which case they may be in large numbers 

and derived from the vagina. A marked increase, as recognized by numerous, 

indistinct, small, circular or irregular, granular cells, should be regarded as 

pathologic.^ To this condition the name pyuria has been given. The simple 

finding of a pyuria does not necessarily indicate the point from which these 

cells were derived. Severe inflammatory processes anywhere along the 

genitourinary tract or the rupture of an abscess into the urinary tract will 

be associated with a pyuria, so that other features must be relied upon in 

deciding as to the source. As a rule, it may be said that the amount of pus 

is small in direct affections of the renal cortex, while disease of the urinary 

passages is associated with a larger number. If an abscess has ruptured 

into the pelvis of the kidney the number of cells may be enormous. If the 

pyuria be of renal origin, it will be associated with the presence of the small, 

round, renal epithelial cells as well as with tubular casts. Frequently leuco- 

^ See Posner, Arch. Diagnosis, 1912, V, 269; also, Pedersen, New York Med. Jour., 1913, 
XCVIII, 1141; MacKay, Northwest Med., 1916, XV, 402. 
24 



370 



DIAGNOSTIC METHODS 



cytes in small numbers are found adherent to the casts or they may even be 
so closely grouped as to give the name pus cast to such formations. If large 
numbers of pus-cells appear in the course of a chronic nephritis, they indicate 
either an acute exacerbation of the condition or a complicating process. in 
some other portion of the urinary tract. The sudden appearance of very 
large numbers of pus-cells is especially indicative of a ruptured abscess. In 
inflammatory processes in the pelvis of the kidney the amount of pus may 
vary within wide limits. In some cases the urine may be perfectly clear when 
voided, showing the presence of only a few pus-cells, while in others enormous 
numbers may appear. This paradoxical condition may be accounted for 




Fig. io6. — Pus corpuscles, i, Normal; 2, showing ameboid movements; 3, nuclei 
rendered distinct by acetic acid; 4, as observed in chronic pyelitis; 5, swollen by ammonium 
carbonate. (Hawk after UUzmann.) 



by the possibility of obstruction of the ureter on the affected side and the later 
forcing out of the large numbers of pus-cells. In pyelitis the urine is usually 
acid, which may serve as a distinguishing point from cystitis in which the 
urine is almost always alkaline. 

In tuberculosis of the renal parenchyma pus-cells appear very early and 
vary in number from a few to many thousands. This pyuria is usually con- 
stant, and is frequently associated with hematuria. The pus-cells in tubercu- 
losis are usually of the monuclear type instead of the ordinary polymorphonu- 
clear form. This is not easily determined, as the degenerative processes make 
it somewhat difficult to distinguish the nuclear form. In such conditions 
the sediment should be frequently examined for the presence of tubercle 
bacilli and a portion inoculated into a guinea-pig. This is the only certain 
method of making a diagnosis of tuberculosis of the kidney. It is, perhaps, 
needless to add that for absolute differentiation a specimen obtained by 
ureteral catheterization must be examined. 

In cystitis the number of pus-cells appearing in the urine will vary ac- 
cording to the severity of the condition, the more severe the more pus-cells. 



THE URINE 371 

In this condition the urine is alkaline and may be, when voided, glairy and 
ropy. In chronic cases of cystitis the pus-cells, although present in the blad- 
der in large numbers, may be so degenerated by the alkahnity of the contents 
that practically no cells are recognizable. Here we find the appearance 
of a large amount of mucus, the urine being in some cases distinctly jelly-like. 

In inflammatory processes of the urethra pus may be present in varying 
amounts. In the acute conditions the number of cells is much more numer- 
ous than in the chronic types. The recognition of the causative factor, in 
most cases the gonococcus, will be treated of in a later section. As the acute 
condition becomes subacute or chronic, the urine contains large numbers of 
the so-called gonorrheal threads which enclose numerous pus-cells.^ These will 
be treated in detail later. It is sometimes a matter of clinical importance to 
distinguish "between an anterior and a posterior urethritis. This is best done 
by the so-called " two-glass " test. If the first portions of the urine be collected 
in a receiving vessel and the later portions in a second vessel, the urine in 
the first vessel will be cloudy while that in the second vessel is clear in the case 
of anterior urethritis ; while in posterior urethritis associated with the anterior 
type the first portion will be cloudy and the second usually so, although at 
times it may be clear. The reaction of the urine in both vessels will be acid 
unless a comphcating cystitis has arisen, when the urine in the second vessel 
will usually be alkaline. 

The appearance of the pus-cells will vary depending upon the reaction 
of the urine. In acid urine their structure is very well preserved, the addition 
of acetic acid rendering the nucleus somewhat more distinct. Their usual 
form is that of the polymorphonuclear neutrophile, their size varying from 
7 to 1 2 microns. If stained- the vesicular character of the nucleus of the renal 
epithelial cell will absolutely differentiate it from the irregular type of pus-cell. 
In alkahne urine the cells swell up, lose their shape and become opaque. 
The addition of acetic acid usually clears them in such a way that the nucleus 
becomes visible, but occasionally does not. If the urine remains long in con- 
tact with the alkaline material in the bladder it becomes slimy, stringy, and 
gelatinous, owing to its large content in mucus. Albumin is always present 
so that it may be difficult to decide whether or not a true albuminuria exists. 

As the pus-cells may undergo such marked change when in alkahne urine 
and be converted entirely into a gelatinous mass in which corpuscles cannot 
be detected, certain tests must be applied for positive recognition of pus in 
such cases. 

VitaU's Test. 

Acidify the urine with acetic acid and filter. Treat the material on 
the filter with a few drops of tincture of guaiac, when a deep blue color will 
appear in the presence of pus. If the material is not filterable, as happens 
when the purulent material is extremely gelatinous, place a portion of this 
slimy urine in a test-tube and allow a few drops of tincture of guaiac to flow 

^ See Broadman, New York Med. Tour., 19 13, XCVIII, 28; also, Thomas, Am. Jour. 
Med. Sc, 1913, CXLVI, 696. 

2 See Fittipaldi, Gaz. d. Osp., 1915, XXXVI, 1265. 



372 DIAGNOSTIC METHODS 

upon the surface. If pus be present a distinct blue line of contact will be 
observed. 

Donne's Test. 

A portion of the urinary sediment in a centrifuge tube is treated with 
a few drops of concentrated solution of sodium hydrate. If pus be present 
an extremely viscid gelatinous mass will be obtained. If this mixture be 
heated, it will dissolve, according to Miiller, with the formation of /?-nucleinic 
acid. 

If the pus-cells be treated under the microscope with a few drops of Lugol's 
solution, they will take a mahogany-brown color owing to the presence of 
glycogen. 

Enumeration of Pus-cells. 

Such a procedure is not a part of the ordinary routine examination of 
urine. It is, however, sometimes advisable, as it permits of a decision regard- 
ing the presence or absence of a true albuminuria. If the latter exists, casts 
and renal epithehal cells will usually be present so that a diagnosis is often 
possible without a count of the cells. 

Technic. 

A portion of the 24-hour specimen of urine is thoroughly shaken to bring 
the corpuscles into suspension. This turbid fluid is then drawn up to the 
upper (11) mark of the leucocytometer, a drop placed upon the glass slide 
and the cells counted as described under Blood. If more than 30,000 per 
cmm. are present, it is advisable to dilute five times with 3 per cent, sodium 
chlorid solution. For each 100,000 leucocytes per cmm. of urine o.i per cent, 
of albumin is assumed to be present, according to Wunderlich. 
(4) Red Blood-ceUs. 

The presence of red blood-cells in the urine is known as hematuria. This 
condition should be sharply differentiated from hemoglobinuria as the clinical 
significance is entirely distinct. Blood may be found in the urine in a variety 
of conditions. Thus in the more malignant types of the acute infectious 
fevers hematuria is frequently observed. Likewise, in scurvy, hemophilia, 
purpura, leukemia, and Werlhof's disease, the kidney may be so markedly 
affected that hematuria obtains. 

In the hematuria of purely renal origin we find both acute and chronic 
congestions as well as inflammatory processes in the kidney associated with 
this condition.^ In the more acute types of nephritis, hematuria is so com- 
mon that the name "hemorrhagic nephritis" is frequently applied. Such 
cases are especially observed after posioning with cantharides and phenol 
derivatives. 2 The chronic parenchymatous type of nephritis is, according to 
Weigert, always hemorrhagic in type, the number of red corpuscles being an 
indication of the intensity of the process. In malignant growths of the kid- 
ney, tuberculosis, renal calculus, and cystic degeneration of the kidney, 
hematuria is especially common; while in infection with certain parasites, 

^ See Randall, Jour. Am. Med. Assn., 1913, LX, 10. 

2 See Belkowski, Rev. de. med., 1913, XXXIII 663, for a discussion of hematuria caused 
by urotropin. 



THE URINE 373 

such as the filaria, echinococcus, and the distoma hematobium, hematuria 
is relatively frequent although few cases of these conditions are seen. 

Hematuria may also be observed as a result of lesions or disease of any 
portion of the urinary tract. Thus stone in the ureter or urethra, tumors, 
ulcers, and parasites of the bladder, urethritis, prostatitis or injury during 
catheterization may also be associated with the appearance of red cells in the 
urine. 

A further type of cases in which hematuria occurs is known as the func- 
tional or idiopathic hematuria. In this class of cases no definite lesion has 
been found to account for the condition. It has been called ''Gull's renal 
epistaxis," "essential renal hematuria," "angioneurotic hematuria," "renal 
hemophilia" and "renal aneurysm." The lesion, whatever it may be, is 
usually unilateral and the attacks appear at variable intervals. Some of the 
cases recover without any treatment after one or two profuse hemorrhages, 
while others require extensive treatment of the nervous system.^ 

In the diagnosis of a hematuria it is important to observe the appearance 
of the urine both with the naked eye and with the microscope. The urine is 
turbid, and varies from a light, hazy, "smoky" appearance to a bright-red 
or deep-brown color. The red cells appear in various stages of preservation. 
In some cases the normal yellow color of the cell will be quite distinct while in 
others the color will be entirely washed out. If the urine be particularly con- 
centrated many crenated forms will also be observed. 

The blood-cells may exist singly and scattered, or may be grouped in large 
masses forming distinct clots or adherent to tube casts forming the so-called 
blood casts. In true renal hematuria the blood is intimately mixed with 
the urine, the individual corpuscles usually appearing as pale shadows or 
"ghosts." In hemorrhage from the bladder the urine may show the presence 
of blood-clots of irregular form and size. If the two-glass test be applied, the 
second glass will contain the more blood, while in hematuria of renal origin 
both glasses will show equal amounts. In some cases clots of blood in dis- 
tinct casts are seen. In the chronic parenchymatous nephritis clots are rarely 
present, while in malignant disease of the kidney clots are relatively commen. 

It is important in making a diagnosis from the presence of blood that 
extraneous sources of blood-cells be excluded. If the blood be of renal origin 
it will be associated with the presence of casts and epithelial cells while no such 
elements will be present from a hemorrhage lower down in the genito-urinary 
tract. Albumin will also be present in more or less amount. It has been 
stated that if the blood be derived from other than renal sources, the clear 
supernatant fluid in the centrifuge tube will be albumin-free. The writer 
has convinced himself that this is an error, as he practically always obtains 
faint albumin reactions in cases of hemorrhage other than renal. 

1 See Braasch, Jour. Am. Med. Assn., 1913, LXI, 936; Kemble, Wash. Med. Ann., 1914, 
XIII, 150; Kretschmer, Interstate Med. Jour., 1914, XXI, 1256; Wulff, Hospitalstid, 1914, 
LVII, 1465; Kornmann, Miinch. med. Wchnschr,, 1915, LXII, 1422; Krotoszyner, Calif. 
State Jour. Med., 1915, XIII, 415; Lipschitz, Cor. Bl. f. Schwerz. Aerzte, 1915, XLV, 
1473; Wildbolz, Ibid., 1586; Sanders, N. Y. Med. Jour., 1916, CIII, 1226; Gast, Deutsche 
med. Wchnschr., 1916, XLII, 1166; Kretschmer, Jour. A. M. A., 1917, LXVIII, 598; 
Wiseman, Am. Jour. Med. Sc, 1917, CLIV, 264. 



374 



DIAGNOSTIC METHODS 



(5) Casts. 

True casts are moulds of the uriniferous tubules. Their mode of forma- 
tion is not entirely clear. Undoubtedly a colloid substance is thrown into the 
lumen of the tubule and later solidifies forming a distinct cast of that particu- 
lar tubule. In this process of hardening the material may enclose cells of 
different types which are, also, present in the tubule. Whether this coagula- 
ble material is derived from the blood as a transudate, whether it be a secre- 
tion of the epithelial cells which have become pathologic, or whether it be 
material arising from degeneration of renal cells is not at present settled, the 
latter source being the more probable.^ In the urine we find true casts of the 
renal tubules, as well as pseudo casts which have nothing in common with the 
true type of these pathologic formations. 

True Casts. 

Hyaline Casts. 

The true hyaline casts are pale, transparent, homogeneous cylinders with 
rounded ends. Their size may vary from a very small fragment to one several 
mm. in length. In diameter they may be narrow or broad. As a rule, little 




Fig. 107. — Hyaline casts. One cast is impregnated with four renal cells. (Hawk.) 

difference is clinically made between these various types of hyaline casts, but 
the broader types seem to the writer to be somewhat more significant than do^ 
the narrower ones. The characteristics of the true cast are their cylindrical 
appearance, their sides being parallel and usually straight, although they may 
at times be observed in typical tortuous forms. They are never tapering at 
the ends, but may show an irregular outline at one or both ends, but the 
length and the parallel sides will usually differentiate them. 
^ See Erdman, Jour. Am. Med. Assn., 1912, LIX, 1952, 



THE URINE 375 

The pure hyaline casts are perfectly homogeneous and free from granules. 
Such types are, however, not frequently observed as very fine granules may 
almost always be detected embedded in the surrounding homogeneous ma- 
terial. There may be even inclusions of epithelial, renal, blood-, or pus-cells^ 
so that the gradations between the pure type of hyaline casts and many of 
the other varieties are outlined with diflGiculty. It should be stated at this 
point that a distinction exists between hyaline casts with enclosures of cells 
to such an extent that the cast is named from the variety of cell present and 
the type of pseudo cast in which groups of such cells are massed so as to form 
an apparent cast, but which do not have any definite matrix. The true hya- 
line cast is soluble in acetic acid and may be stained yellow withLugol's solution. 

These hyaline casts are not always easy to find in the sediment. In 
examining the urine for the presence of casts the light should be shut off as 
much as possible and a low-power lens u^ed. With the use of the high power 
the field is limited and one is not so apt to observe the cast as with the low 
power. It is, however, always advisable to examine a cast, first seen with the 
low power, under the high power, so that the decision may be much more 
definite as to whether the cellular elements are real inclusions or simply 
material resting upon the true cast. The same is to be said regarding the 
presence of granules in the hyaline cast. 

Significance. 

Regarding the significance of hyaline casts in the urine, it is to be said that 
they occur in any condition in which the kidney is altered by circulatory,, 
toxic, or inflammatory disturbances. They are not pathognomonic of any 
one condition and may be found as a result of simple functional disturbance. 
A few hyaline casts may be found in practically every urine, providing suffi- 
cient search is made. Any undue strain, such as running for a car in one 
who is not used to such exertion, may be sufficient to add quite a number of 
hyaline casts to the urine. In many thousand urine examinations made in the 
writer's laboratory, it has been rather the unusual thing not to find an occa- 
sional hyaline cast. It would seem, therefore, that no significance whatever 
should be attached to the presence of an occasional hyahne cast. When,, 
however, these casts become very numerous they should then be interpreted 
as meaning a disturbance of the kidney, although the absence of other types 
would rather speak against a marked pathologic change. In diabetes melli- 
tus, "showers" of casts are especially observed preceding the appearance of 
coma (Kiilz). 

Granular Casts. 

These are modifications of the true hyaline cast in the sense that fine or 
coarse granules are found in the matrix of the hyaline cast. Several types of 
granular casts are observed. The granules may be very fine, very coarse, or 
may be distinctly composed of denegerated epithelial cells. The fine as well 
as the coarse granules are undoubtedly derived from the renal epithelium, 
which has degenerated completely. The coarser the granules the more severe 
the inflammatory process. 



376 



DIAGNOSTIC METHODS 



These granular casts vary in shape and in size, but are usually shorter 
than the hyaUne type. To these granular casts may be attached various 
cells so that it is difficult to tell whether the cast is really a true granular or a 
cellular one. In some cases these cellular inclusions may undergo fatty 
degeneration giving a much higher refractiUty to the^specimen. Not infre- 
quently one observes hyaUne casts which are distinctly granular in one 
portion while the other is perfectly homogeneous. _ 




Fig. io8. — Granular casts, X 225. {Tyson.) 

The so-called hrown granular casts appear to be almost entirely degener- 
ated epitheUal cells, although the coloring matter is probably hemoglobin. 
The fact that the hyaline matrix cannot be distinctly made out does not argue 
against this type being truly hyahne in character, although the matrix is 
completely saturated with the pigment. 

Waxy Casts. 

This type is very refractile, transparent, and either perfectly colorless or 
showing a sHght shade of yellow. Usually they are very long and broad and 
may be either straight or curved. The ends show a very distinct fracture 
while the cast itself may show a tendency to spUt transversely. Their ap- 
pearance is, therefore, that of ordinary wax. They may have any t3^e of 



PLATE XL 




U 



Waxy Casts Treated with Iodine. {Tyson) 



THE URINE 



377 



cellular element attached and may show marked fatty degeneration. Some 
of these casts show the amyloid reaction while many of them do not. 

Waxy casts were at one time believed to be pathognomonic of amyloid 
degeneration of the kidney. It is true that they do appear earlier in this 
type of kidney lesion, but it is to be remembered that they occur in all 
varieties of chronic kidney disease. They are usually of bad prognostic 
omen as they indicate a very advanced process. 

Fibrinous Casts. 

These are very highly refractile, transparent, and always of a yellowish 
or brown color. They may be granular and have various cellular inclusions. 
Their shapes vary as do those of the hyaline types and they show a tendency 
to become fractured, the fracture usually being ragged, 
while in the waxy cast it is sharp-cut. 

Fibrinous casts usually appear in the acute renal 
conditions and disappear when this condition clears 
up. They do not have, therefore, the grave signifi- 
cance of the waxy cast and should be sharply differen- 
tiated. These casts are not composed of fibrin as their 
name would indicate, but are so called on account of 
their brownish color, which resembles fibrin. 

Epithelial Casts. 

These casts are true hyaline casts which include 
so many renal epithelial cells that the hyaline matrix 
may be lost. For the name epithelial cast to be ac- 
curate it is not necessary that more than a few cells 
be present. The cells may be well preserved or show 
marked fatty or granular degeneration. The nuclei 
of these cells are round and vesicular so that they may be easily recognized. 

Distinct gradations exist between the true epithelial cast and the coarsely 
granular and fatty cast. This type of cast is indicative of a severe destructive 
lesion of the kidney epithelium. 

Fatty Casts. 

These casts are masses of epithelial cells which have so markedly degener- 
ated that little is recognized beyond the original outline of the cell and the 
numerous fatty globules contained therein. They may be yellowish or black 
in color, the globules being soluble in ether and staining black with osmic acid 
or red with Sudan III. 

Blood Casts. 

These are casts including large numbers of blood-cells. The casts 
are formed within the tubules of the kidney, the cells occasionally being 
very pale. These casts indicate a serious advanced lesion of the renal 
parenchyma. 

Pus Casts. 

These like the other types of casts are true hyaline casts with enclosures 
of pus-cells. They are formed within the tubules of the kidney and usually 




Fig. 109. — E p i t h e 1 i a 1 

casts. {Hawk.) 



378 



DIAGNOSTIC METHODS 



indicate an acute pyelonephritis. For the differentiation of these casts from 
epithehal casts it is advisable to add acetic acid to the sediment when the 
typical polymorphous character of the nucleus will distinguish the pus-cell 
from the epithelial cell with its vesicular nucleus. Moreover, the pus-cell is 
much more spherical thaii is the epithelial cell. 

Cylindroids. 

It is not infrequent to find in the urine formations which resemble the 
true hyaline casts to a marked degree. They, however, differ in the fact that 
at one or both ends they taper off into a point which may be prolonged into 
a distinct thread. If, however, these ends are broken off, as may occur in the 
centrifugation, it is a practical impossibility to distinguish them from a 
hyaline cast. They are both found in the urine under the same conditions 
and their significance is practically the same. From the chemical standpoint 




Fig. iio. — Fatty casts. {Hawk after Peyer.) 



they appear similar to the hyaline casts, their origin, therefore, being pre- 
sumably in the renal parenchyma. If these bodies are true mucin and are 
insoluble in acetic acid, their origin is more probably in the bladder. 

A second type of cylindroid appears in the urine in the form of long taper- 
ing transparent shreds. They very much resemble ribbon which varies in 
diameter and may show under high power a distinctly fibrillar structure. 
These threads largely compose the nubecula. They are much longer than the 
hyaline cast and considerably narrower so that confusion should not arise. 
In cases of gonorrhea one finds mucous shreds which may vary from a few 
mm. to I cm. in length and yellowish or pure white in color. In the meshes of 
these shreds one finds embedded large numbers of pus and epithelial cells. 
These should be sharply differentiated from the true cast by their larger size 



PLATE XII. 




Mucous Threads in Urine. (Unstained Specimen ) 



iki 



THE URINE 



379 



and typical mucoid character. Frequently they may be observed by the 
naked eye in large numbers. 

Pseudocasts. 

Not infrequently do we find in urine crystalline material arranged in 
masses much resembling casts. The most important of these are uric acid 
and the urates. It is true that any cast in a concentrated urine may become 
covered with urates so that the true nature of the cast becomes indefinite. 
If the slide be warmed these pseudourate casts will disappear, while the true 
casts will remain. Masses of bacteria, pus-cells, epithelial cells and blood- 
cells may so group themselves as closely to resemble true casts. As a rule 
such masses will show irreg.ular outlines and no evidence of a distinct matrix. 
Moreover, the use of an old slide upon which there may be many scratches 




Fig. III. — Blood, Pus, Hyaline and Epithelial Casts. {Greene.) 

a, Blood casts; 6, pus cast; c, hyaline cast impregnated with renal cells; 

d, epithelial casts. 

should be avoided as the writer has seen several instances in which supposed 
casts were found to be due to such scratches. 

Cylindruria. 

This is the name given to the appearance of casts in the urine. As a rule, 
it should be said that the presence of a few hyaline casts is not of particular 
moment unless associated with other evidences of marked renal disturbances. 
While albuminuria and cylindruria usually go hand in hand, yet we do find 
cases in which one exists without the other. 

It is undoubtedly true that casts indicate a disturbance of the renal epithe- 
lium. This, however, need not be anything more than disturbed nutritional 
or circulatory conditions. However, when the true hyaline casts are present 
in large numbers and when many other types of casts also exist, then a dis- 



38o 



DIAGNOSTIC METHODS 



tinct pathologic lesion of the kidney must be assumed.^ As a rule, the gran- 
ular types of cast are observed in the chronic processes, while the cellular 
forms are more usually present in the acute conditions. This rule, however, 
is not invariable, so that it may generally be stated that no type of cast is 
pathognomonic of any single condition. In this connection we should re- 
member that recent work, especially that of Cabot, has shown that it is un- 
wise to base a diagnosis of a kidney lesion upon the finding even of both 
albuminuria and cylindruria. So much discrepancy was shown to exist be- 
tween the urinary and autopsy findings that one must remain in doubt as to 
whether it is possible to make a definite diagnosis unless clinical symptoms 
other than urinary are made the basis of a diagnosis. On the other hand, 




Fig. 112. — Cylindroids. {Hawk after Peyer.) 

some of the most typical cases of nephritis, as shown postmortem, gave ab- 
solutely no indication in the urine that such condition existed. We are, 
therefore, face to face with the proposition that urinary examination must be 
in any case simply one of the diagnostic links. This fact is of special import- 
ance in life insurance examinations, as most companies absolutely refuse 
insurance to one who has ever shown albumin or casts in the urine. This 
would seem to the writer not only very short sighted, but based upon an 
absolutely erroneous idea of the importance of albumin and casts in the urine 
of one who showed absolutely no clinical signs of renal involvement. Re- 
membering that albumin and casts may not appear, even though the kidney 
be seriously affected, it would seem just as plausible to refuse life insurance 
because these substances were not present. In this connection the writer 
would say that only when the urine is considered as a whole may definite 

^ See Barringer and Warren, Arch. Int. Med., 1912, IX, 657; also, Posner, Berl. klin. 
Wchnschr., 1913, L, 2040; Minerbi, Policlinico, 1914, XXI, 1667. 



THE URINE 381 

conclusions be made regarding any type of renal disease. The clinician, who 
is thoroughly familiar with the course of the case, is the only one capable of 
interpreting the findings of the laboratory, so that it should be an unvarying 
rule for a laboratory worker to avoid diagnostic remarks unless he is thor- 
oughly en rapport with the patient. The writer does not wish to be inter- 
preted as stating that a diagnosis of renal disease may never be made from an 
examination of the urine, but he wishes to impress upon his readers that both 
albuminuria and cylindruria may occur without direct kidney disease or may 
not appear when such is present. 

(6) Spermatozoa. ., 

Spermatozoa are frequently observed in the urine of healthy adults, 
especially after intercourse or nocturnal emissions. In females they may also 
be observed as an evidence of intercourse, which fact is of some importance in 
cases of suspected rape. 

Pathologically, they may be found in cases of marked constipation, when 
the pressure of the impacted feces upon the seminal vesicles may induce an 
emission. In occasional cases of cystitis, associated with stricture, these 
bodies may be observed as reported by Simon. In cases of epilepsy and 
hysteroepilepsy, as well as in spinal disease following vertebral fractures and 
dislocations, spermatozoa are not infrequent. Masturbation and venereal 
excess frequently lead to almost constant spermatorrhea. Their occurrence in 
cases of prostatitis will be discussed in the section on Semen. 

(7) Tissue Fragments. 

It is not infrequent to find shreds of tissue in the urine, which may throw 
some light upon a pathologic condition. In cases of carcinoma of the bladder, 
more rarely of the kidney, true malignant tissue may be obtained, which may 
permit of a tentative diagnosis, although the material is usually too necrotic 
to make an absolute diagnosis possible. 

(8) Bacteria. 

It should be stated in the beginning of this discussion that an examination 
of the urine for bacteria should be made only upon specimens obtained with 
the greatest possible precaution and preserved in absolutely sterile vessels^ 
Soon after the urine is voided, especially if it remains in contact with the air,, 
large numbers of saprophytic organisms may be found which, of course, did 
not exist in the original urine. In obtaining a specimen from the male it is 
not always necessary to catheterize the patient. If the surface of the glans 
and the orifice of the meatus be carefully washed with bichlorid solution 
followed by sterile water and the first portion of the urine voided be thrown 
away, the last portion may be collected in a sterile vessel and later put in 
work. With female patients, however, it is absolutely essential that cathe- 
terization be performed. The external genitaha and especially the orifice- of 
the urethra are well washed with green soap and water. The opening of the 
urethra is then dried with sterilized cotton pads which are soaked in boracic 
acid. A sterilized glass catheter, whose external end is covered with a rubber 
tube about four inches long and large enough to fit loosely over the catheter is 

^ See Hort, Jour. Hyg., 1914, XIV, 509. 



382 DIAGNOSTIC METHODS 

then inserted, care being taken that it touches only the orifice of the urethra. 
The urine is allowed to flow freely for a short time when the last portion is 
collected in a sterile vessel, the rubber tube being previously removed 
(Kelly). Cultures are then made from the urine and the remainder centri- 
fuged in a sterile closed tube in order to throw down any bacteria which may 
be present. It is sometimes advisable, in order to diminish the specific 
gravity of the specimen, to add an equal volume of 95 per cent, alcohol and 
centrifuge the mixture. Practically all of the bacteria present will then be 
found in the 'sediment. 

The supernatant fluid is then removed by quickly inverting the tube and 
allowing the fluid to run out. The sediment, by this manipulation, will usually 
remain in the smaller portion of the tube. Smears are then made upon a glass 
slide and dried first in the air and then over the flame. It is not always the 
simplest matter to prepare smears which will remain after treatment with the 
staining solution, as the urea and salts of the sediment may be removed by the 
washing and carry with them the bacteria. If pus-cells are present, satis- 
factory smears are usually obtained; but if such conditions do not exist it is 
advisable to add a solution of egg albumin to the sediment before drying over 
the flame. 

The methods of staining the sediment for the various bacteria will depend 
entirely upon the organism supposed to be present. As a rule, a preliminary 
examination is very satisfactorily made by treatment with Loffler's methy- 
lene-blue solution, which stains practically all organisms. If the tubercle 
bacillus is suspected it may be detected in exactly the same way as outlined 
under Sputum by staining with carbol-fuchsin solution. Should one suspect 
the presence of the gonococcus^ this may be stained, as described in the next 
section, by Gram's method. Outside of these two types of organisms, it is 
almost impossible to differentiate the bacteria of the urine by staining 
methods. 

If the urine be collected with the precautions mentioned above, any organ- 
isms found must be attributed to their presence in the urine as voided. In 
this connection we must remember that the presence of the tubercle bacillus 
does not necessarily indicate tuberculosis along the genitourinary tract. ^ 
Tubercle bacilli are found in the urine in cases of mihary tuberculosis and 
have been reported in pulmonary tuberculosis, although it is more frequent to 
find them as evidences of local tubercular conditions. Of course, if large 
numbers of pus- and blood-cells be present along with tubercle bacilli, the 
diagnosis is usually certain. A word of caution is, however, necessary at this 
point. The smegma bacillus grows in abundance on the external genital 
organs and its morphological and staining characteristics may closely re- 
semble those of the tubercle bacillus.^ If the proper precautions be observed, 

^ This must not be confused with the micrococcus catarrhalis, which not-infrequently is 
the etiologic factor in genitourinary inflammations. 

2 See Kielheuthues, Folio urol., 191 2, VII, 191; Beer, Am. Jour. Med. Sc, 1917, CLIV, 
251; Humbert, Rev. Med. de la Suisse Rom., 1917, XXXVII, 35; Watson, Am. Jour. 
Med. Sc, 1918, CLVI, 636; Spooner, Jour. Med. Res., 1918, XXXIX, 59. 

3 See Gautier, Jour. d'Urol., 1914, V, 161; Brereton and Smith, Am. Jour. Med. Sc, 
1914, CXLVIII, 267; Churchman, Ibid., 722; Editorial, Jour. Am. Med. Assn., i9i5,LXIV, 
348; Brown, Ibid., 886. 



THE URINE 383 

as they should be, no differentiation is necessary, bacilU showing the true 
morphological and staining character of the tubercle bacillus can be only this 
organism, as extraneous bacteria have been avoided. In the clinical labora- 
tory, however, one may never be sure whether the proper precautions have 
been taken, so that absolute methods of differentiation (see Sputum) should 
be part of the technic unless the worker absolutely knows that contamination 
was avoided. A second point regarding the tubercle bacillus is that it may 
not be found by microscopic examination even after repeated attempts. 
Under such conditions the wisest course to pursue is the inoculation of a 
guinea-pig with the washed urinary sediment. The obtaining of the urine 
must in this case be absolutely accurately done by observing every precaution 
to prevent contamination. The reason for this is not because contaminating 
organisms will cause lesions similar to those of the tubercle bacillus, but be- 
cause such secondary invaders may so infect the animal that death results 
from causes other than those for which we are looking. The washed sedi- 
ment is injected intraperitoneally and the animal kept under observation for 
three weeks to one month unless death results previously.^ At the end of this 
time the animal is killed and a postmortem examination made for evidences 
of tuberculosis. The retroperitoneal glands, spleen, and liver are the especial 
organs to show such lesions. These organs should be sectioned, portions run 
through the regular pathologic routine, and sections examined microscopic- 
ally. A finding of tuberculosis in this way is unequivocal and is the quickest 
way in the long run of making a positive diagnosis, although a single examina- 
tion may show the presence of tubercle bacilli in the urine, but rarely such 
is the case.^ 

Having found the tubercle bacilli in the urine, we are confronted with the 
question of the part affected. As a primary tuberculosis of the bladder is rare 
we may usually assume the seat of the difficulty to be the kidney, although if 
evidences of cystitis be present a combination may exist. As the symptoms 
of genitourinary tuberculosis are frequently vesical in origin, a kidney lesion 
may not be suspected, but should be assumed until the contrary is proven. 
Thanks to the introduction of methods of cystoscopic examination and espe- 
cially ureteral catheterization, we are in a better position to make a positive 
diagnosis of renal tuberculosis and exclude that of bladder origin. An in- 
teresting point regarding tuberculous cystitis is that the urine, although 
frequently containing large amounts of pus, is practically always acid in re- 
action. Moreover, this pus is frequently sterile in tubercular cystitis. It is 
not the province of the writer, at the present time, to outline methods of 
differential diagnosis of various conditions; he will refer, therefore, to works 
on genitourinary diseases for the various types of cystitis and their clinical 
differentiation. Suffice it to say at this point that cystitis may be an ascend- 
ing or a descending one and should always be correlated with the associated 

^ Morton (Jour. Exper. Med., 1916, XXIV, 419) has shown that the use of guinea pigs, 
which have been exposed to X-rays, permits a diagnosis in from 8 to 10 days. 

2 See Bryan, New York Med. Jour., 1913, XCVIII, 20; also, Keene and Laird, Am. Jour. 
Med. Sc, 1913, CXLVI, 352. 



384 



DIAGNOSTIC METHODS 




condition.^ It is usually a simple matter to determine the presence of the 
gonococcus in the urethral discharge, but it is far from easy to demonstrate 
this organism in the case of gonorrheal cystitis. Under such conditions the 
symptoms and the association with an existing gonorrhea would furnish the 
decisive clue. 
Bacilluria. 

By this condition is meant the presence in the freshly voided urine of so 
many organisms that the urine is distinctly cloudy. These organisms are 
usually those associated with a mild cystitis or may be those of an existing 
general infection. ^ In cases of persistent bacilluria, we may find a true renal 
origin, which is largely associated with the presence of the typhoid and colon 
bacillus. As has been well established, the typhoid organism may be excreted 
for months after the patient is convalescent so that it becomes necessary to 
use strict measures to disinfect all urine of typhoid patients. The colon 

bacillus is at present assuming so much 
importance in clinical work that cases are 
being almost daily recognized which may 
be directly traceable to the colon bacillus 
and not to the typhoid as usually assumed. 
It is necessary, therefore, for the labora- 
tory worker especially, and, where possi- 
ble, for the general practitioner to be able 
to recognize each of these organisms when 
present either in the feces, urine, milk, 
or water-supply (see Feces). A second 
general class of cases associated with 
bacilluria are those of urethritis and pros- 
tatitis. Usually there is a secondary 
cystitis arising from the same organism 
or, at least, the resistance of the bladder has been so far reduced that these 
organisms which find their way into the bladder develop profusely therein. 
(9) Parasites. 

Various types of parasites are observed in the urine. Thus the tricho- 
monas vaginalis has been found by Kiinstler, Miura, and Dock. Amebae 

1 See Peterkin, Urol, and Cut. Review (Tech. Supp.), 1915, III, 188; Beeler and Helm- 
holz, Am. Jour. Dis. Child., 1916, XII, 345; Mathers, Jour. Infect. Dis., 1916, XIX, 416; 
Curtis, Jour. A. M. A., 1916, LXVI, 1456; Dick and Dick, Arch. Int. Med., 1917, XIX, 
493; Thomas, Am. Jour. Med. Sc, 191 7, CLIII, 701; Schwartz, Am. Jour. Dis. Child., 191 7, 
XIII, 420; Eisendrath and Schultz, Jour. Med. Res., 191 7, XXV, 295; Jour. A. M. A., 
1917, LXVIII, 540; Crabtree and Cabot, Ibid., 589; Quinby, Ibid., 591; Coyon and Lemi- 
erre. Bull. Soc. Med. Hop. Paris, 1919, XLIII, 743. 

2 Freifeld (Berl. klin. Wchnschr., 1913, L, 1761) reports the finding of diphtheria bacilli 
in urine. Rosenow (Jour. Infect. Dis., 1909, VI, 296) and Townsend (Jour. Am. Med. 
Assn., i9i3,LXI, 1605), report the pseudo-diphtheria bacillus as a causative factor of cystitis. 
See, also, Curtis, Jour. Am. Med. Assn., 1915, LXIV, 270; Surg., Gyn. and Obs., 1915, 
XXI, 423. Barber and Draper, Jour. Am. Med. Assn., 1915, LXIV, 205; McGowan, Ibid., 
226; Cunningham, Ibid., 231; Koll, Ibid., 297; Greeley, New York Med. Jour., 1915, CI, 
250; Hoover, Interstate Med. Jour., 1915, XXII, 163; Kretschmer, Ibid., 173; Dick, Dick 
and Rappaport, Jour. Inf. Dis., 1916, XVIII, 216; Morishima and Teague, Ibid., 1917, 
XXI, 145; Herrold and Culver, Ibid., 1919, XXIV, 114; Schmidt, III. Med. Jour., 1919, 
XXXVI, 188 and 241. 



^^ 



Fig. 113. — Scolex and booklets of 
taenia echinococcus in urine. 



PLATE XIII. 






y-Tit •-• / 






i 



Cystitis due to Colon Bacillus. (Methylene Blue Stain.) 



PLATE XIV 



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f^ 



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STAPHYLOCOCCrS CYSTITIS. (LeISHMAN 



Stain.) 



THE URINE 



38s 



have been found by Balz, Jlirgens, Wijchoff, and by Musgrave and Clegg. 
Various portions of hydatid cysts are frequently observed, among which we 
find the echinococcus booklets and fragments of membrane. Nematode 
worms, especially the filaria sanguinis hominis, are present in cases of chyluria, 
while the anguillula aceti or " vinegar- eel" has been reported, especially by 
Stiles, while Billings and Miller report its presence as a possible contamina- 
tion from the bottle in which the urine was collected.^ 

Eggs of the schistosomum haematobium are not infrequently observed 
together with large numbers of red cells in cases of bilharziasis. This worm 
as well as its ova will be discussed in the section on Blood. Stuertz has 
reported the findings of the egg of eustrongylus gigas in the urine in a case 
of chyluria. 

V. Calculi 

Concretions of a more or less hard and dense character are prone to form 
in the urinary passages. These bodies are termed, according to their size 
and location, sand, gravel, stone, and calculi. These formations consist of 
accretions of any of the various crystalline or amorphous sediments previously 
mentioned, the type of stone depending upon the reaction of the urine. 

C 






Fig. 114. — Ova and miracidium of schistosomum hematoblum, X 300: A, Ovum as seen in 
urine; B, the same after addition of water; C, miracidium. (Tyson after Railliet.) 

These calculi are formed by the deposition of the crystalline material 
around a definite nucleus, which usually consists of organic material, such as 
fibrin, blood, desquamated epithelial cells, mucus, or even a crystal of uric 
acid or calcium oxalate. ^ It is very difficult to decide as to the reason for the 
deposition of this material in the form of a renal stone. The growth of the 

^ 1 King (Jour. Am. Med. Assn., 1914, LXIII, 2285) reports a case of Myiasis of the 
urinary passage due to the presence of the larva of the small "latrine fly" (fannia scalar is). 
2 See Posner, Ztschr. f. Urol., 1913, VII, 799; also, Lichtwitz, Ibid., 810; also, Williams, 
New York Med. Jour., 1915, CII, 609; Woolley, Jour. Lab. and Clin. Med., 1916, I, 848; 
Smith, Boston Med. and Surg. Jour., 191 7, CLXXVI, 524; Young, Ibid., 1919, CLXXXI, 
573. 

25 



386 DIAGNOSTIC METHODS 

calculus takes place by accretion, the deposition of successive layers of mate- 
rial occurring around the original nucleus. The material of which the stone 
consists will usually be of one kind, so that we speak of uric acid or phosphate 
calculi, for instance, while, occasionally, mixed calculi may be formed by the 
deposition of two or more chemical combinations. 

The classification of urinary concretions is based on the chemical constit- 
uents of which they are composed. Before examining a calculus chemically 
a thorough optical examination should be made, as this may give a definite 
clue as to its composition. After this preliminary examination, the calculus 
is ground to a fine powder and examined according to the following table of 
Heller. 

Uric Acid Calculi. 

These are, perhaps, the most common renal stones. They are not always 
composed of pure uric acid, but are made up of a mixture of this substance 
with the urates. They are always colored, usually yellowish or brownish, 
but may at times appear distinctly red. They are fairly hard and usually 
show a rough irregular nodular surface, although at times this may be smooth. 
They fracture very easily and show, on cross section, a distinctly laminated 
structure, the layers frequently being of different colors, in some cases even 
being composed of deposits other than uric acid. If heated on a platinum 
foil they are combustible, burning without a flame. They give the murexid 
test and do not liberate appreciable amounts of ammonia on treatment 
with sodium hydrate. 

Ammonium Urate Stones. 

The ammonium urate calculi occur rarely in the adult. They are small, 
yellow and very soft, being distinctly clay-like and easily powdered when 
dry. These stones give the murexid test and also give a strong reaction 
for ammonia on treatment with sodium hydrate. 

Calcium Oxalate Stones. 

Next to the uric acid calculus the oxalate stone is most frequently met.^ 
The smaller types of these calculi are practically colorless and have a smooth 
surface, while the larger ones are grayish, brownish or even black in color 
and have a rough nodular surface with sharp projecting angles. These 
stones frequently cause severe hemorrhage and much irritation when passing 
through the ureter and urethra. From their appearance they have been 
called the mulberry calcuH. They are, perhaps, the hardest of the urinary 
stones. 

These calcuH are insoluble in acetic acid, but soluble in hydrochloric 
acid without effervescence unless the powder is previously heated. It is rare 
to find these stones perfectly pure, admixtures with various other sediments 
leading to distinct concentric arrangement as shown on fracture. 

1 See Rowlands, Biochem. Jour., 1908, III, 346; Mackarell, Moore and Thomas, Ibid., 
1910, V, 161; Kahn and Rosenbloom, Jour. Am. Med. Assn., 1912, LIX, 2252; also, Kahn, 
Arch. Int. Med., 1913, XI, 92; Kahn, Ztschr. f. exp. Path. u. Therap., 1915, XVII, 88; 
Rosenbloom, Jour. y\m. Med. Assn., 1915, LXV, 161; Cabot, Surg. Gyn. and Obs., 1915, 
XXI, 403; Braasch and Moore, Jour. Am. Med. Assn., 191 5, LXV, 1234. 



THE URINE 



387 



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3^8 DIAGNOSTIC METHODS 

Phosphatic Calculi. 

Stones composed of pure alkaline phosphates or triple phosphates are 
exceedingly rare. Usually the phosphatic calcuH contain admixtures of 
ammonium urate, calcium carbonate, and calcium oxalate. The color of 
such stones may range from a white through yellow to some with distinct red- 
dish tones. They are frequently of very large size, especially when formed 
in the bladder, are of a chalky consistency, and show a rough surface. 

These calcuH are soluble in hydrochloric or acetic acid, such solutions 
giving reactions both for phosphoric acid and the alkahne earths. 

Calcium Carbonate Calculi. 

Such stones are exceedingly rare. They are small in size, are distinctly 
chalk-like in consistency and color, and have a smooth surface. If treated 
with acid, carbon dioxid is evolved, 

Cystin Calculi. 

These stones are white or pale yellow in color, have either a smooth 
or irregular surface, and are soft and waxy in consistency. They vary in 
size occasionally being found as large as a hen's egg, although those of true 
renal origin are about the size of a pea. The formation of such calculi and 
their passage through the ureter and urethra constitute practically all of the 
untoward symptoms shown by subjects affected with cystinuria.^ 

Such stones burn readily if heated on a platinum foil, giving off a peculiar 
sharp odor. The powder is soluble in ammonia from which the characteristic 
hexagonal plates separate on allowing the ammonia to evaporate. 

Xanthin Calculi. 

These stones occur especially in children, although even here they are very 
rare. They are usually light brown in color, moderately hard, and vary in 
size from that of a pea to a tennis-ball. On cross section they appear amor- 
phous and if rubbed take a polish much resembling that of wax. The pow- 
der shows the typical reaction for xanthin previously outlined. ^ 

Urostealith Calculi. 

These masses consist of fat, calcium and magnesium soaps, and choles- 
terin. They are usually soft and may be somewhat irregular in shape. This 
material burns with a pale yellow flame giving an odor of resin. The "dry 
powder is soluble in alcohol and ether, from which rhombic notched plates of 
cholesterin separate on evaporation. 

VI. Functional Diagnosis 

It is usually of great importance, especially in cases in which surgical 
intervention is contemplated, to know just exactly what the functional capa- 
bilities of the kidney are. If one kidney is to be removed, the question arises 
as to whether the remaining kidney can sufficiently accommodate itself to the 
increased work which must be put upon it. 

1 See Frankenthal, Deutsch. Ztschr. f. Chir., 1914, CXXXI, 442; Neumann, Deutsch. 
med. Wchnschr., 1914, XL, 2065; Abderhalden, Ztschr. f. physiol. Chem., 1919, CIV, 129. 

2 See Rosenbloom, New York Med. Jour., 1915, CI, 120. 



THE URINE 389 

As such a variation has been occasionally found between the results of the 
chemical and microscopical examination of the urine on the one hand and the 
pathologic condition shown in the kidneys postmortem on the other, an at- 
tempt has been made to find delicate tests by which a true renal lesion might 
be indubitably determined and thus permit of an absolute diagnosis even 
though the urinary findings were or were not conclusive. It is granted that 
a more or less severe lesion of the kidney may exist and yet its functional 
capacity be almost normal. If this functional capacity can be determined by 
tests which are more or less simple, it is evident that such methods should 
form part of the daily routine of the practitioner.^ 

The methods of functional renal diagnosis fall, in a general way, into 
four groups. 

1. The determination of the rate of" excretion in the urine of a known 
amount of a chemical substance, injected or ingested. Especially note- 
worthy in this group is the method of Rowntree and Geraghty, which will be 
discussed in detail. Other tests^ along this line, are those advanced by 
Schlayer and Takayasu^ of the intravenous administration of 20 c.c. of a 10 
per cent, solution of lactose or the administration, per os, of 0.5 gram of po- 
tassium iodid and the determination of the time and degree of elimination. 
These latter tests are not, in the writer's opinion, as reliable as that of 
Rowntree and Geraghty and will be omitted, although they are worthy of 
trial in doubtful cases. 

2. The comparison of the excretion of substances normally eliminated, 
as water, salt, and nitrogenous bodies, with the amount of intake of such 
substances in a test diet. This method has been especially elaborated by 
Mosenthal and will be freely discussed. Similar, although by no means as 
reliable, are the urea and salt excretion tests, following the administration 
of a known quantity of these substances. 

3. The determination of the extent of retention in the blood of normal 
metabolic products, especially of nitrogenous substances. This phase of 
the renal tests will be discussed in detail in the section on Blood. 

4. A determination of the ratio between the concentration of urea in the 
blood and its excretion in the urine. The results are expressed in terms of 
Ambard's coefficient or of McLean's Index. These will be taken up in the 
section on Blood. 

It is to be mentioned that no one test of renal function is to be regarded 
as a standard lor all cases. So many factors enter into the consideration of 
this question, that "to speak of testing renal function as a whole is to set a 
false standard." It is only when all the available data is before one, that 
proper deductions may be made. This has been excellently expressed by 

1 See Blum, Renal Diagnosis in Medicine and Surgery, New York, 1914; Stevens, Jour. 
Am. Med. Assn., 1914, LXII, 1544; Geraghty, New York Med. Jour., 1914, C, 312; Piersol, 
Interstate Med. Jour., 1914, XXI, 1165; Thomas, Jour. Am. Med. Assn., 1914, LXIII, 
1909; Braasch and Thomas, Ibid., 1915, LXIV, 104; Geraghty, Bull. Johns Hopkins Hosp., 

1915, XXVI, 155. 

2 Deutsche Arch. f. klin. Med., 1911, CI, 2>2>y'> see, also, von Noorden, Med. Klin., 

1916, XII, 5; Treupel, Deutsche med. Wchnschr., 1916, XLII, 155; Falk and Sugiura„ 
Jour. Pharm. and Exper. Therap., i9i7;|IX, 241. 



39° DIAGNOSTIC METHODS 

Mosenthal and Lewis in the following words: "Of the various tests for 
renal function, each has its own significance, and a greater insight will be 
obtained into the characteristics of kidney disease when physicians will no 
longer advocate one test blindly to the exclusion of all others, but will 
endeavor to interpret each one according to its significance." 

Cryoscopy. 

The method of determining the freezing-point of a solution is one of 
the most delicate of those of physical chemistry. As it is based upon the 
principle that substances in solution lower the freezing-point of the solvent in 
direct proportion to the molecular or ionic concentration of the solution, this 
method serves as a ready means of determining the molecular weight of a 
substance as well as the molecular concentration of a solution. For a successful 
outcome of a cfyoscopic determination, the most assiduous attention to detail 
must be paid, so that this method certainly can find no place in the hands of the 
general practitioner or even in those of the laboratory worker, who has not 
been especially trained along these lines. Such being the case, it is absurd 
to expect that slight variations in the freezing-point (A) of such a complex 
mixture as the urine can yield any valuable information, especially when 
one remembers that fluctuations wider than those shown under pathological 
conditions may be noted as a result of not observing such slight details as that 
of constantly agitating the urine during the cooling and freezing. The 
normal freezing-point of the urine varies between — 0.9 and — 2°C., an increase 
being known as hypersthenuria and a decrease as hyposthenuria. 

As will be seen in the discussion of this subject in the section on Bloody 
the results obtained by this method have been far from satisfactory, as noth- 
ing of distinct diagnostic value has as yet been derived from comparative 
studies by various workers.^ It would seem to the writer, therefore, that for 
the present this method would better be left to the research worker than to be 
adopted by the general or special student, who should make use of methods 
which will yield results of more immediate value to him. For these reasons 
the writer must refer to other works for a detailed description of the method. 

Electric Conductivity. 

The remarks made later in the section on Blood regarding electric conduc- 
tivity are especially applicable to the urine. This test is altogether too deli- 
cate to be appUed to such complex fluids as the urine with the hope that 
slight variations in the conductivity will show anything of importance. As 
the electric current is conducted only by dissociable compounds, this method 
can show absolutely nothing regarding the excretion of the nondissociable 
organic substances. Such being the case the writer can see no reason for re- 
sorting to such delicate procedures as the determination of the conductivity 
of the urine when no attempt is made, as may be observed in many of the ex- 
periments reported, to control the intake of the inorganic substances which 
would affect the conductivity of the urine. A little more attention to ordi- 
nary methods of chemical examination, with especial regard to ascertaining 

1 See Desgrez, Bull. soc. pharmakolog., 1915, XXII, 159. 



THE URINE 391 

the intake and output of the patient, would, in the writer's opinion, yield 
much more valuable information than could be obtained by the rather un- 
certain urinary manipulations with the method of Kohlrausch. 
Mosenthal's Test Meal for Renal Function. 

In 1 9 14 Hedinger and Schlayer^ proposed a test for estimating renal 
function based upon the response of the kidney, as shown by its output, to 
an intake of a dietary containing considerable quantities of diuretic sub- 
stances such as fluids, salts, and purins. The various influences, which sod- 
ium chlorid, for instance, may exert upon the excretion of water had been 
previously shown by Mosenthal and Schlayer.^ Before administering their 
test diet, Hedinger and Schlayer placed the subject for the three days preced- 
ing the test upon a reasonable general diet, which contained, as necessary 
constituents, 8 to 12 grams of salt and about 2000 c.c. of water. If this 
amount of fluid was deemed too large, a smaller amount might be used but 
the amount on each of the three days should be the same. This preliminary 
attention to diet is deemed by these authors of importance in establishing 
a definite fixed output of water and salt. On the day of the test, the patient 
is directed to empty his bladder completely at 7 a.m. and is then started on 
his *'test meal," which is divided into 5 meals at different hours throughout 
the day. The urine is collected at two-hour intervals until 9 p.m., when the 
total voiding from that hour until 7 a.m. is collected in one container. In 
each specimen of the day urine and in the total night urine the specific 
gravity, total volume and amount of NaCl are determined. O'Hare^ has 
followed this method in a study of the renal function in nephritis. 

In 1 91 5 MosenthaP introduced a modification of this method, which, 
with the further changes suggested by him in 191 8, is the method followed 
in performing this very valuable and important test. The directions for 
this test meal are taken from his original article and comprise the instructions 
given to the nurse in charge of the case. 

All food is to be salt free food from the diet kitchen. 

Salt for each meal will be furnished in weighed amounts. (One capsule 
of salt, containing 2.3 grams of sodium chlorid, is furnished for each meal. 
The salt which is not consumed is returned to the laboratory, where it is 
weighed and the actual amount of salt taken is calculated.) 

All food or fluid not taken must be weighed or measured after meals and 
charted in the spaces below. 

Allow no food or fluid of any kind except at meal times. 

Note any mishaps or irregularities that occur in giving the diet or collect- 
ing the specimens. 

Breakfast, 8 a.m. 

Boiled oatmeal, 100 gm. 
Sugar, 1-2 teaspoonfuls 
Milk, 30 c.c. 
Two slices bread (30 gms. each) 

1 Deutsch. Arch. f. klin. Med., 1914, CXIV, 120. 

2Ibid., 1913, CXI, 217. 

3 Arch. Int. Med., 1916, XVII, 711. 

^Ibid, i9i5,XVI, 733. 



392 DIAGNOSTIC METHODS 

Butter, 20 gms. 

Coffee, i6c c.c. 

Milk, 40 c.c. 

Sugar, I teaspoonful 

Milk, 200 c.c. 

Water, 200 c.c. 
Dinner, 12 Noon. 

Meat soup, i8c c.c. 

Beefsteak, 100 gms. 

Potato (baked, mashed or boiled), 130 gms. 

Green Vegetables, as desired 

Two slices bread (30 gms. each) 

Butter, 20 gms. 

Tea, 180 c.c. 

Sugar, I teaspoonful 

Milk, 20 c.c. 

Water, 250 c.c. 

Pudding (tapioca or rice) no gms. 
Supper, 5 P.M. 

Two eggs, cooked in any style 

Two slices bread (30 gms. each) 

Butter, 20 gms. 

Tea, 180 c.c. 

Sugar, I teaspoonful 

Milk, 20 c.c. 

Fruit (stewed or fresh) i portion 

Water, 300 c.c 

The patient empties his bladder at 8 a.m. (the beginning of the test meal), 
the specimen being discarded. The urine is collected in separate containers 
at two-hour intervals until 8 p.m. The total voiding from 8 p.m. to 8 a.m. 
is collected in one container and constitutes the night urine. 

This dietary contains approximately 13.4 grams of nitrogen, 8.5 grams of 
salt, 1760 c.c. of fluid and a considerable quantity of purin material in the 
meat, soup, tea, and coffee. All these substances act as diuretics, and it is 
on the mode of excretory response to such stimuli that the test of renal 
function depends. 

In the earlier studies with this test, the above dietary was followed, but 
it has become questionable whether such a rigid adherence to it is necessary, 
as such practice makes it difficult to follow in private work. In his later 
article MosenthaP has studied this test under the influence of 3 different 
dietaries, namely, the above, which he calls his "high protein diet;" a ''low 
protein diet," which is the usual one consisting of some such combination 
as the following: 

Breakjast. 
Sherry (30 c.c), baked apple, stewed prunes, orange, hominy cornstarch 
(J^ hominy, }:3 cornstarch), cereal, cream (15 c.c), sugar, butter. 

Dinner. 
Sherry (30 c.c), potato (baked or mashed), string-beans, cabbage, car- 
rots, lettuce, onions, tomatoes, cucumber pickles, fruit cornstarch pudding, 
tapioca pudding, sugar, butter. 

1 Arch. Int. Med., 1918, XXII, 770. See, also, Mosenthal and Lewis, Jour. A. M. A., 
1916, LXVII, 933; Desha, Southern Med. Jour., 1916, IX, 1041; Christie, Jour. Lab. and 
Clin. Med., 1917, II, 899; Moses, Med. Record, 1917, XCI, 273; Vaughan, Jour. Lab. and 
Clin.' Med., 1918, III, 531; Veer and Saunders, Jour. A. M. A., 1919, LXXII, 1586. 



THE URINE ^ 393 

Supper. 

Same as dinner. 
No restrictions being placed on the intake of this diet, the nitrogen equivalent 
is about 3 to 4 grams. The third diet studied by Mosenthal was styled the 
"normal diet," which consisted of the food which the patient chose for 
himself. Specimens were collected as in the test with the '' high protein diet," 
outlined above, the only exception being that if the night meal was later than 
5 P.M. the collection of the night specimen began three hours after this 
meal. The results of these studies is that the response of the kidney is not 
dependent, as far as the interpretation of this test is concerned, on the diet, 
so that the value of the test is widely extended to general private practice. 

As in the original test of Hedinger and Schlayer, the volume and specific 
gravity of each specimen collected are determined. The salt and nitrogen 
may be determined in each specimen, but all that is necessary is to make 
such determinations in the total day and total night urines. 

In the earlier work with this test, the standards set as normal have been 
shown to be, to a certain extent at least, erroneous, chiefly owing to neglect 
in considering the extra-renal factors which influence the output of the kid- 
ney. "Nervous tension, very hot or cold weather, and what may be termed 
the 'renal habits' of any person, all have appeared to play a distinct role in 
causing differences to appear that were not at fi.rst appreciated." To these 
conditions we must add the effect of the state of the water reserve of the 
tissues and the chilling of the body surface, as shown by Lyle and Sharit.^ 
These authors state "These factors can function significantly to distort the 
test meal reaction when viewed in the terms suggested by Mosenthal. When 
so functioning they affect the fluid element in the test meal reaction; and this 
mainly by virtue of the fact that the skin and lungs make a preferential and 
significant demand on body fluids, w^hereas the excretion of solids by the 
skin and lungs is practically negligible." 

The normal standard, as originally advocated by Mosenthal, was the 
following: A maximum specific gravity of 1018 or higher; a variation of 9 
degrees or more between .the minimum and njaximum specific gravity read- 
ings; and a night urine of 400 c.c. or less, with a specific gravity of 1018 or 
over and a concentration of nitrogen of at least i per cent.; the balance be- 
tween the output and intake of salt, nitrogen, and fluids should be approxi- 
mately equal. For the reasons mentioned in the previous paragraph, these 
standards can no longer be regarded as correct. The newer standards, as 
advanced by Mosenthal, represent the present conceptions of the renal 
esponse to this test, the chief changes being noted in the characteristics of 
the night urine. Under the influence of a high or a low protein diet or of the 
normal diet, the maximum specific gravity is to be regarded as 1018 with the 
high diet and 1020 with the low or normal diets. A variation of 9 points be- 
tween the high and low specific gravity should obtain. With the normal 
diet, the degrees of variation in the specific gravity readings is of no value. 
The specific gravity of the night urine has no significance in this new standard. 

1 Arch. Int. Med., 1918, XXI, 366. 



394 DIAGNOSTIC METHODS 

The volume of the night urine should be 750 c.c. or less. The nitrogen and 
sodium chlorid in the night urine or the highest per cent, in any specimen 
is regarded as normal if i per cent, or higher, but as not necessarily abnormal, 
if less. 

When kidney function becomes involved, the first signs are usually 
demonstrated in the night urine, the quantity increasing. While the specific 
gravity and nitrogen concentration may become lowered, yet this is not 
necessarily true, so that these factors are no longer regarded as important. 
Marked variations may be brought about in the elimination of edema fluids, 
so that the change from oliguria to polyuria may appear with rapidity and 
influence the interpretation of this test. In severe cases of nephritis, an 
advanced degree of functional inadequacy of the kidney is indicated by a 
markedly fixed and low specific gravity, a diminished output of both salt and 
nitrogen, a tendency to total polyuria and a night urine showing an increased 
volume, with, usually, a low specific gravity and low concentration of nitro- 
gen. As Mosenthal states, these functional pictures are found regularly in 
many other conditions, as pyelitis, cystitis, hypertrophied prostate, marked 
anemia,^ pyelonephritis, polycystic kidney and diabetes insipidus. In 
chronic diffuse nephritis, the condition of renal function is characterized by 
its variabiHty. The findings in myocardial insufficiency vary according 
to the activity of the heart. This test gives the earliest indication of dim- 
inished kidney efiiciency and, likewise, reaches the maximum degree of 
impairment before the other functional tests. A careful study of this is 
recommended. 

Phloridzin Test. 

This test is based on the assumption that phloridzin normally gives rise to 
a glycosuria through distinct alternations in the renal cells. In other words, a 
so-called "renal diabetes" is set up. The technic is as follows: i c.c. of a 
1 :2oo aqueous solution of phloridzin is injected subcutaneously. The urine is 
tested at intervals of 15 minutes for the appearance of sugar. Normally, 
sugar may be detected in one-half to one hour and may be present for as long 
as five hours. The quantity ehminated may vary from 0.5 to 3 grams of glu- 
cose. In nephritis the sugar is usually absent or below 0.5 gram. The test 
does not distinguish the various types of nephritis, yet it does usually indi- 
cate that renal activity is disturbed. It is, however, being rapidly displaced 
by the following test. 

Phenoisulphonephthalein Test. 

Rowntree and Geraghty^ have introduced a test for the functional activity 
of the kidney which is by far the most accurate at our disposal.^ The solu- 
tion used is prepared as follows: 0.6 gram of phenoisulphonephthalein and 

1 Christian, Ibid., 1916, XVIII, 429. 

2 Jour. Pharm. and Exper. Therap., 1910, I, 579; Arch. Int. Med., 1912, IX, 284» 

^ See Conzen, Deutsch. Arch. f. klin. Med., 191 2, CVIII, 353; Smith, Am. Jour. Dis. 
Child., 1913, V, 25; Pepper and Austin, Am. Jour. Med. Sc, 1913, CXLV, 254; also, Rown- 
tree, Fitz and Geraghty, Arch. Int. Med., 1913, XI, 121; Geraghty, Rowntree and Gary, 
Am. Surg., 1913, LVIII, 800; Dietsch, Ztschr. f. Exper. Pathol, u. Therap., 1913, XIV, 512; 
Rowntree, Geraghty and Marshall, Surg. Gynec. and Obs., 1914, XVIII, 196. 



THE URINE 395 

0.84 ex. of 2/N NaOH solution (8 per cent.) are added to sufficient 0.75 per 
cent. NaCl solution to make 100 ex. Each e.e. of this solution contains, 
therefore, 6 mg. of the dye. As the mono-sodium salt, formed in this solu- 
tion, is slightly irritant locally when injected, 2 or 3 drops more of the 2/N 
NaOH are added, when the solution changes to a beautiful Bordeaux red 
color and becomes non-irritant.^ 

The technic is as follows: 20 to 30 minutes before applying the test, the 
patient is given 300 to 400 e.e. of water in order to insure a free urinary secre- 
tion, otherwise delayed time of appearance may be due to lack of secretion. 
Under aseptic precautions a catheter is introduced into the bladder and the 
bladder completely emptied. Noting the time, i c.c. of the above solution is 
administered subcutaneously in the upper arm by means of an accurately 
graduated syringe. The urine is allowed to drain through the catheter into a 
test-tube in which has been placed a drop of 25 per cent. NaOH solution and 
the time of the first pinkish tinge is noted. In patients without urinary 
obstruction, the catheter is withdrawn at the time of appearance of the drug 
in the urine and the patient is instructed to void into a receptacle at the end of 
one hour and into a second receiver at the end of the second hour. A rough 
estimate of the time of appearance may be made by having the patient void 
urine without the use of the catheter at frequent intervals. In prostate cases 
it is wise to have the catheter in place until the end of the observation. The 
catheter is corked at the time of appearance of the drug in the urine and 
the cork is removed at the end of the first and second hours, the bladder 
being drained each time. 

Each sample of urine is measured and the specific gravity taken. Suffi- 
cient 25 per cent. NaOH is added to make the urine decidedly alkaline in order 
to elicit the maximum color (a brilliant purple-red). This solution is now 
placed in a liter volumetric flask and distilled water added to the i L. mark. 
Thoroughly mix the solution and filter a small portion for comparison with the 
standard solution. This standard consists of 3 mg. of phenolsulphone phtha- 
lein (or i c.c. of the solution used for injection) diluted to i liter and made 
alkaline with i or 2 drops of 25 per cent. NaOH solution. This solution will 
retain its intensity of color for weeks. The comparison of color is made in 
the Duboscq or the Rowntree and Geraghty modification of the Autenrieth- 
Konigsberger colorimeter. The known (standard) solution is adjusted to the 
10 mm. mark and the intensity of the unknown solution is made to correspond 
by means of the usual manipulations. One may readily calculate the amount 
of drug eliminated as follows: If, for instance, the reading for the unknown 
solution is 20, it is evident that only 50 per cent, as much dye is present as is 
contained in the standard solution. That is, the excretion equals 50 per cent, 
of the 3 mg. of the standard or 25 per cent, of the 6 mg. injected. The read- 
ings may be then made on a second and third voiding until the drug is com- 
pletely eliminated. 

- 1 This mono-sodium salt of phenolsulphonephthalein may be obtained from Hynson, 
Wescott and Co. of Baltimore in the form of ampules, each c.c. of the solution containing 
6 mg. 



396 DIAGNOSTIC METHODS 

In normal cases it has been found that the time of appearance varied from 
5 to 1 1 minutes and that 40 to 60 per cent, of the drug was excreted in the first 
hour and 20 to 25 per cent, in the second hour. The excretion of the drug 
does not run parallel to the excretion of water. The smaller the amount of 
urine in normal cases the greater the concentration of the drug. It is im- 
material, as far as the excretion of the drug is concerned, whether the urinary 
output is 50,200,400 or more c.c. 

In pathological cases it has been demonstrated that the permeability of 
the kidney for this drug is decreased in both chronic parenchymatous and 
chronic interstitial nephritis, the decrease being most marked in the latter and 
varying with the intensity of the disease. In cases with obstruction in the 
lower urinary tract, this test may show renal involvement to such an extent 
that operation should be deferred, especially if the time of appearance is de- 
layed beyond 25 minutes and the output of the drug is below 20 per cent, for 
the first hour. This point is of special value to the surgeon as it shows the 
danger of using surgical intervention until the kidney may become less in- 
sufficient. It would seem to be of value, also, in the study of the renal con- 
dition preceding uremia. To the obstetrician it may show the possible 
approach of eclampsia, as has been demonstrated in several cases. In uni- 
lateral and bilateral kidney diseases the absolute amount of work done by 
each kidney as well as the relative proportion can be determined by resort 
to ureteral catheterization followed by this test. The writer has no hesitancy 
in advising a careful study of this test in all cases with suspected or actual 
renal involvement.^ 

BIBLIOGRAPHY 

1. Benedict. Influence of Inanition on Metabolism. Washington, 1907. 

2. Blarez. L'Urine au point de vue clinique et medical. Paris, 1906. 

1 See Behrenroth and Frank, Ztschr. f. exper. Path. u. Therap., 19 13, XIII, 72; Rowntree 
and Fitz, Arch. Int. Med., 1913, XI, 258; Geraghty, Jour. Am. Med., Assn., 1913, LX, 191; 
Goodman, Ibid., 1913, LXI, 184; Frothingham, Fitz, Folin and Denis, Arch. Int. Med., 

1913, XII, 245; Geraghty and Rowntree, Jour. Am. Med. Assn., 1913, LXI, 939; Foster, 
Arch. Int. Med., 1913, XII, 452; Fishbein, Jour. Am. Med. Assn., 1913, LXI, 1368; Roth, 
Berl. klin. Wchnschr., 1913, L, 1609; Marogna, Gazz. d. osp., 1914, XXXV, 172; Widal, 
Weill and Radot, Presse med., 1914, XXII, 565; JPezzana, Policlinico, 1914, XXI, 563; Mach- 
witz, Rosenberg and Tschirtkoff, Munch, med. Wchnschr., 1914, LXI, 1268; Hess, Ibid., 
1835 and 1874; Ware, New York Med. Jour., 1914, XCIX, 416; Robertson, Ibid., 972; 
Jones, Ibid., 1914, C, 518; Fanz, Ibid., 1214; Tracy, Surg., Gyn. and Obs., 1914, XIX, 734^ 
Block, Jour. Am. Med. Assn., 1914, LXII, 1309; Griessmann, Deutsch. Arch. f. klin. Med., 

1914, CXIV, 32; Deutsch and Schmuckler, Ibid., 61; Hedinger and Schlayer, Ibid., 120; 
Hessel, Ibid., 396; Lichtwitz and Stromeyer, Ibid., 1914, CXVI, 127; Rowntree, Am. Jour. 
Med. Sc, 1914, CXLVII, 352; Fitz, Ibid., 1914, CXLVIII, 330; Thayer and Snowden, 
Ibid., 781; Agnew, Arch. Int. Med., 1914, XIII, 485; Frothingham and Smillie, Ibid., 

1914, XIV, 514; Quimby and Fitz, Ibid., 1915, XV, 303; Miller and Cabot, Ibid., 369; 
Rowntree, Marshall and Baetjer, Ibid., 543; Hopkins and Jones, Ibid., 964; Bauer and 
Habetin, Ztschr. f. Urol., 1914, VIII, 355; Bauer and Nyiri, Ibid,, 1915, IX, 81; Theston 
and Comfort, Am. Jour. Dis. Child., 1915, X, 278; Hess, Bull. Johns Hopkins Hosp., 

1915, XXVI, 52; Smith, Jour. Am. Med. Assn., 1915, LXIV, 223; Elliott, Ibid., 1885, 
Pedersen, New York Med. Jour., 1915, CI, 770; Siegel, Pub. Jefferson Med. Coll., 1915; 
VI, 78; Hempelmann, Am. Jour. Dis. Child.,. 1915, X, 418; Naroditzky, Russk. Vrach, 

1915, XIV, 850; Lubs and Acree, Jour. Am. Chem. Soc, 1916, XXXVIII, 2772; Barker 
and Smith, Am. Jour. Med. Sc, 1916, CLI, 44; Brown and Cummins, Jour. A. ]\I. A., 

1916, LXVI, 793; Peabody, Boston Med. and Surg. Jour., 1916, CLXXV, 158; Cameron, 
Jour. A. M. A., 1916, LXVI, 1765; Kendall, Ibid., i9i7,LXVIII, 343; Arbuthnot, Snowden 
and Brooks, Jour. Pharm. and Exp. Therap., 191 7, IX, 349. 



THE URINE 397 

3. Blumenthal. Pathologic des Harnes am Krankenbett. Berlin, 1903; Hand- 

buch der Speziellen Pathologic des Harns. Berlin, 1913. 

4. Chittenden. Physiological Economy in Nutrition. New York, 1905. The 

Nutrition of Man. New York, 1907. 

5. CoHNHEiM. Chemie der Eiweisskorper. Braunschweig, 1906. 

6. Croftan. Clinical Urinology. Chicago, 1907. 

7. Daiber. Mikroskopie der Harn-Sedimente. Wiesbaden, 1906. 

8. Fischer. Untersuchungen ueber Aminosauren. Berlin, 1906. 

9. Gerard. Traite des Urines. Paris, 1906. 

10. Gerard. L'analyse des urines consideree comme un des elements de diagnostic. 

2* ed. Paris, 19 13. 

11. Hammarsten. Lehrbuch der physiologische Chemie. Wiesbaden, 19 14. 

12. Heitzmann. Urinary Analysis and Diagnosis. New York, 19 15. 

13. Hoppe-Seyler-Thierfelder. Handbuch der physiologisch- und pathologisch- 

chemischen Analyse. Berlin, 1909. 

14. Krehl. Ueber die Storung chemischer Korrelationen im Organismus. Leipzig, 

1906. Pathologische Physiologic. Leipzig, 1912. 

15. MACLEOD. Physiology and Biochemistry in Modern Medicine, 1919. 

16. Mann. The Physiology and Pathology of the Urine. London, 1913. 

17. Naunyn. Der Diabetes Mellitus. Wien, 1907. 

18. Neubauer und Huppert. Analyse des Harns. Wiesbaden, 1910 and 1913. 

19. Neuberg. Der Harn. Berlin, 191 1. 

20. von NooRDEN. Die Zuckerkrankheit. BerHn, 1912. 

21. Ogden. Clinical Examination of the Urine. Philadelphia, 1903. 

22. Pavy. Carbohydrate Metabolism and Diabetes. London, 1906. 

23. PuRDY, Practical Urinalysis and Urinary Diagnosis. Philadelphia, 1900. 

24. Rieder AND Delephine. Urinary Sediments. London, 1899. 

25. Saxe. Examination of the Urine. Philadelphia, 1906. 

26. Scott. Clinical Examination of the Urine. Philadelphia, 1900. 

27. Spaeth. Untersuchung des Harns. Leipzig, 1903. 

28. Sutton. Volumetric Analysis. Philadelphia, 1904. 

29. Tyson. Bright's Disease and Diabetes. Philadelphia, 1904. 

30. Wells. Chemical Pathology. Philadelphia, 191 4. 



CHAPTER VII 
SECRETIONS OF THE GENITAL ORGANS 

I. Male Secretions 

General Considerations. 

The normal secretion of the male generative organs is known as semen 
and is a mixture of the secretions of the prostate gland, the glands of Cowper, 
the testicles, and the seminal vesicles. It is a practical impossibility from the 
clinical standpoint to separate the different elements of the semen, so that 
this must be discussed as a whole. 

The semen is a white or slightly yellowish, somewhat thick and viscid 
liquid with a peculiar odor, somewhat resembling fresh glue, and showing a 
neutral or faintly alkaline reaction, a nonhomogeneous milky appearance, and 
a specific gravity greater than that of water. It is composed of semisolid 
material in the form of white masses floating in a limpid liquid and holding 
in suspension specific elements, derived from the secretory glands of the geni- 
tal apparatus and from the desquamation of the various canals through 
which the semen passes. 

Semen contains about 6 per cent, of organic and 4 per cent, of inorganic 
matter. Its chief chemical characteristic is the presence of spermin (C2H5- 
N)2, which is at least isomeric, if not identical, with diethylen-diamin, accord- 
ing to Ladenburg and Abel. This spermin, which is derived largely from the 
prostate gland, combines with the phosphoric acid radical to form spermin 
phosphate, which crystallizes in the form of four-sided spindles or prisms 
which may appear as flattened needles. In some cases these crystals re- 
semble very closely the diamond-shaped double pyramids known as 
Charcot-Leyden crystals, which are found in the sputum. They are, how- 
ever, of a different crystalline group and are soluble in formalin, while those 
found in the sputum are insoluble in this menstruum. These spermin 
crystals are known as Bottcher's crystals. Miescher has studied the com- 
position of the heads of the spermatozoa and has been able to isolate 
certain bodies, known as protamins, which are the simplest type of protein 
material.^ 
Microscopic Examination. 

The most important and characteristic constituent of semen are the sper- 
matozoa.^ These sexual elements consist of an anterior broader portion or 
head and a narrow thread-like tail. The former is oval or egg-shaped and 
measures about 5 microns in length, 4 in breadth, and 2 in thickness. Just 
behind this pyriform head is a short cylindrical portion measuring 6 microns 

1 See Steudel, Ztschr. f. physiol.Chem., 1913, LXXXIII, 72. 

2 Koessler (Trans. Chic. Path. Soc, 191 2, VIII, 280) has shown that the spermatozoa 
may act as direct carriers of infectious agents, among these being the spirochseta pallida. 

398 



SECRETIONS OF THE GENITAL ORGANS 



399 



in length, which is known as the middle piece. This tapers somewhat to the 
point of union with the tail. This so-called tail is a thread-like posterior 
portion and is approximately 45 microns in length. In the freshly voided 
semen these tail portions show active undulatory whip-like motions, which 
persist for 24 to 48 hours, and even longer under proper conditions,^ and enable 
the spermatozoa to progress from point to point. Alkalies seem to favor this 
movement, while dilute acids inhibit it very rapidly. This movement^ of the 
spermatozoa is closely associated with their sexual activity, as the cells show- 
ing no movement when freshly voided may be regarded as possessing no 




Katharine- Hill. 

Fig. 115. — Normal semen. 

functional power. For a discussion of spermatogenesis as well as of fertili- 
zation of the ovum the writer would refer to the admirable description of 
McMurrich.^ 

Besides these characteristic portions of the semen, large numbers of 
lecithin globules are seen, which give the milky appearance to the fluid. Espe- 
cially to be noted among the cellular elements are the so-called corpora 
amylacea which resemble very closely starch granules, having concentric 
striations, a finely granular center and occasionally a nucleus. These cells 
take a distinct blue color on treatment with iodin solution. Moreover, 

^ See Ochi, Acta Scholae Med. Univ. Imp. in Kioto, 1916, I, 341; Sato, Ibid., 361. 

^ The statement is frequently made that spermatozoa are motionless till they get be- 
yond the epididymis, but Hiihner (Jour. A. M. A., 1917, LXVIII, 1340) calls attention to 
the motility of the spermatozoa on the testicular side of the epididymis. 

3 The Development of the Human Body, Philadelphia, 1907. 



400 DIAGNOSTIC METHODS 

various epithelial cells are observed, which are derived from the several 
glands contributing to the composition of the semen. Some of these cells are 
distinctly granular, some contain fat globules and very closely resemble the 
colostrum corpuscles of the lacteal secretion, while some of the granules re- 
semble myelin. In rare cases cylindrical casts are seen which simulate the 
hyaline casts of the urine, but as a rule they are larger and longer. These 
are supposed to be derived from the prostate gland and seminal vesicles. If 
the semen be allowed to stand for a few minutes, several types of crystal 
may be observed, especially the phosphate of spermin, ammonium-magne- 
sium phosphate, fatty acids, and oxalate of calcium. This last crystalline 
component is especially noted in the urine in cases of spermatorrhea. 
Pathologic Variations. 

Direct pathologic variations in the semen are limited to two conditions. 
Either spermatozoa are absent or those present are nonmotile. The deter- 
mination of the presence of spermatozoa in the semen or in suspected stains is 
a matter of simple microscopic examination. With the question of the mo- 
tility of such elements, when present, the conditions under which the exami- 
nation is made may markedly influence the findings. If possible, semen 
should be examined, with regard to the motility of the spermatozoa, as soon 
as ejected, but if such is not possible the fluid must be kept warm until exami- 
nation may be made. It is absolutely out of the question to make a positive 
diagnosis of true nonmotility of spermatozoa from examination of specimens, 
which have been allowed to cool. In some cases, if the time has not been too 
extended, warming may bring back the motile power of these cells, but in 
many cases it does not. It is, therefore, unjust and unwise to brand a man 
as sterile without absolute proof that such a condition really exists. If no 
spermatozoa are present, especially after several examinations, sterility is 
absolute. This condition is known as azoospermatism. According to 
Kehrer, 40 per cent, of cases of conjugal sterility are due to the absence of 
spermatozoa in the semen. It is, therefore, necessary that the ordinary 
gynecological idea that women are the responsible factors in the family 
sterility should be, at least, partly borne by the man, as it is unjust to the 
woman to blame her for faults existing in the husband. The writer does not 
wish to be interpreted as stating that sterility does not frequently exist in 
women, but he does desire to emphasize the point that many more men are ster- 
ile than is generally supposed and that the sterile women are in this condition 
largely through the results of gonorrheal infection through their husbands.^ 

Spermatozoa may be absent from the semen during convalescence from 
acute febrile conditions, valvular heart disease, and in general conditions 
associated with lowered nutrition. On the other hand, the constant presence 
of spermatozoa in the urine, as well as in the semen, may be noted as a result 
of various pathological conditions as well as of venereal excesses or mastur- 
bation. To this condition is given the name of spermatorrhea. 

^ See Reynolds, Jour. Am. Med. Assn., i9i3,LXI, 1363; Ibid., 1915, LXV, 1151; Dozzi, 
Gazz. d. osp., 1914, XXXV, 2069; Polak, Surg. Gyn. and Obs., 1916, XXIII, 261; Rey- 
nolds, Jour. A. M. A., 1916, LXVII, 1193; Lespinasse, Ibid., 191 7, LXVIII, 345; Reynolds, 
Ibid., 1919, LXXIII, 1099. 



SECRETIONS OF THE GENITAL ORGANS 401 

Medicolegal Aspects. 

Not infrequently the physician is called upon to decide whether certain 
stains are due to spermatic fluid or whether assault has been committed. If 
the question is one of suspected rape, an examination of a drop of the vaginal 
fluid or of scrapings from the vulva or vagina will usually reveal the sperma- 
tozoa. Of course other signs, which are important from the medicolegal 
point of view, will be observed in the examination of the external organs. 

The stains usually subjected to medicolegal examinations for the presence 
of spermatic fluid have a grayish-yellow color, their size is somewhat variable, 
their contour usually irregular, and the linen upon which the stain is usually 
found is almost as stiff as if it had been starched. As spermatozoa are very 
resistant to the action of reagents as well as to putrefactive processes, they 
may be detected many years after the stain was made. It is, therefore, 
almost an impossibility to say by examination of a stain anything about the 
length of time the stain has been upon the cloth. 

A fragment of the linen, which show§ the stain, is placed in a watchglass 
and allowed to soak for one hour in 30 per cent, alcohol or in faintly alkaline 
water. ^ It is then lifted from the solvent, placed in another watchglass, and 
teased with needles in a solution of i per cent, eosin in glycerin. A few drops 
of this mixture are then placed upon a glass slide, covered with a cover-glass 
and examined with a high-power dry lens. Spermatozoa, if present, will 
practically always be seen by this method. The heads are stained a deep red 
while the tails, which are usually broken off by the teasing, show a light 
reddish tint, which distinguishes them from the unstained vegetable fibers. 

Florence's Test.^ 

It not infrequently happens that spermatozoa may not be found, although 
the stain be due to spermatic fluid. The principle of this test is that sper- 
matic fluid when treated with a solution of iodin in potassium iodid gives 
crystals which were supposed to be iodospermin. According to Bocarius, this 
substance is not iodospermin, but an iodin compound of cholin. This test 
would be given, therefore, by any substance containing cholin and cannot, 
for this reason, be distinctive for spermatic fluid. Such being the case, a 
negative result is of far greater importance than is a positive one, although 
in old stains spermatozoa may be found along with a negative Florence 
reaction. 

Technic. 

The reagent used consists of 2.54 grams of iodin and 1.65 grams of po- 
tassium iodid dissolved in 30 c.c. of distilled water. If a drop of spermatic 
fluid or of an aqueous extract of a suspected stain be treated with a drop of 
this solution and immediately examined under the low-power lens, long 
rhombic platelets of a dark brown color, fine needles, lance-shaped or ovoid 

^ If the stain be relatively fresh, the bit of cloth may be cut out, placed in a watch glass 
and treated with dilute salt solution for two or three hours, the specimen being covered 
with a beaker to prevent evaporation of fluid and to protect it from dust. A drop of the 
turbid fluid is then examined under the microscope. Roussin advises the use of a solvent 
and stain consisting of i part of iodin, 4 parts of potassium iodid and 100 parts of water. 

- Arch. d'AnthropoL, 1896, X, 417 and 520; Ibid., XI, 37, 146 and 249. 
26 



402 DIAGNOSTIC METHODS 

bodies^ often grouped in rosettes may be observed. A positive reaction is 
seen many years after the formation of the stain so that a positive result is of 
value when other sources of cholin are excluded. 

Barberio's Test. 

Barberio has found that the treatment of a drop of spermatic fluid or an 
aqueous extract of a suspected stain with a saturated aqueous solution of 
picric acid gives immediately a precipitate of sharply refractile, yellow, ovoid 
or needle-shaped crystals which gradually increase in size. This test was 
supposed to be of much greater diagnostic importance than that of Florence, 
but the recent work of Fraenkel and Mliller has shown that the crystals are 
not sufficiently characteristic to permit of an absolute diagnosis. They call 
attention to the fact that substances other than spermatic and prostatic 
fluids may give similar crystals, but that in such cases these crystals are iso- 
lated and form usually on the border of the drop, while with spermatic fluids 
the crystals are numerous and are formed throughout the specimen. These 
workers recommend this test for the recognition of prostatic secretions or for 
the condition of azoospermatism, but caution the worker against making an 
absolute diagnosis from its presence in medicolegal cases. A negative result 
does not necessarily exclude the presence of semen. ^ 

It will be seen, therefore, that neither one of the microchemical tests 
given above should be regarded as absolutely indicative of the presence of 
semen. It is much better practice to make repeated search for spermatozoa 
than absolutely to identify a stain as semen by the microchemical method. 



11. Female Secretions 

(i) Vaginal Secretions. 

The normal secretion of the vagina is scanty, usually just sufficient to 
moisten the mucous membrane. It is clear or occasionally opalescent, semi- 
liquid in character, and is composed largely of mucus and epithelial masses. 
Its reaction appears variable. As a rule, it should be considered acid in the 
case of virgins, while in those who have borne children it is usually alkaline. 

Little is known regarding the chemical properties of this secretion. From 
the clinical standpoint the normal vaginal secretion is of importance owing 
to the fact that it possesses marked bactericidal properties. According to 
Kronig, pus organisms introduced into the vagina of pregnant women dis- 
appear in from four to thirty-six hours. Whether this bactericidal power 
is due to the reaction of the secretion or to some unknown agent is unsettled. 
A remarkable fact seems to be that frequent irrigation of the vagina with 
water or antiseptic solutions decreases the bactericidal power. If this be 
true, it is questionable whether frequent douching is advisable.^ 

' See Lecha-Marzo and Conejero, Semana Med., 1913, XX, 74; also Maestre and 
Lecha-Marzo, Ibid., 1914, XXI, 776; Vaughan, Jour. Lab. and Clin. Med., 1916, II, 195. 

2 See Lecha-Marzo, Arch, internat. de Med. leg., 1913, IV, 341; Harada, Am. Jour. Med. 
Sc, 1916, CLII, 243. 

3 See Zweifel, Monatsschr. f. Geburtsh. u. Gynak., 1914, XXXIX, 459. 



SECRETIONS OF THE GENITAL ORGANS 403 

'Microscopic Examination. 

Outside of the great number of large, irregular, stratified, squamous 
epithelial cells which are constantly found in the vaginal secretion, one 
observes mucous corpuscles, a few large mononuclear leucocytes, cellular 
debris, and numerous bacteria. The bacterial fiora^ of the vagina is very 
extensive. These organisms are normally saprophytic and rarely take on 
pathologic functions, but they may occasionally do so. Among these bacteria 
we find the colon bacilH, streptococci, staphylococci, and bacilli which are not 
unlike true diphtheria bacilli. None of these organisms are particularly im- 
portant from the clinical standpoint and will be passed with mere mention. ^ 
We do find, however, certain organisms which give rise to no particular clini- 
cal symptoms, but which are extremely confusing in the examination for the 
presence of the gonococcus. As will be learned from the later discussion of 
this organism of Neisser, it appears in the form of biscuit or coffee-berry 
shaped diplococci, which are both intra- and extracellular and do not stain by 
Gram's method. In the early cases the intracellular organisms are few in 
number. Saprophytic cocci often have the same morphology as the gonococ- 
cus, but are slightly larger, are variable to Gram's stain, and are rarely 
intracellular. They are, however, differentiated by the fact that they grow 
easily upon ordinary media, while the gonococcus requires special media. 
The more or less normal presence of these saprophytes should be constantly in 
mind and a diagnosis of gonorrhea made only when clinical symptoms are 
present to point to the gonococcus. It is wise in all doubtful cases to resort 
to cultivation, as one may very much regret having made a diagnosis of 
gonorrhea when such did not really exist. The practitioner should be cau- 
tioned to take his smear high up, in the vagina, preferably from the region of 
the cervix uteri. If this be done as a routine fewer specimens will be found 
showing these confusing saprophytes. Smears are frequently sent to labora- 
tories for examination which will not show the gonococcus even though pres- 
ent in large numbers in the vagina. It is not sufficient to take a specimen 
from simple purulent material which may be present in the lower portion of 
the vagina as the organisms are frequently absent in these locations. 
Pathology. 
Blennorrhea. 

Physiologically, an increased vaginal secretion (blennorrhea) is seen dur- 
ing sexual excitement, preceding menstruation, and during pregnancy, when 
a very profuse secretion may be observed. If this secretion contains a large 
number of epithelial cells and leucocytes, as seen in nonspecific inflammations, 
it becomes more or less creamy in color and is then called leucorrhea.^ This 
type of leucorrhea should be sharply differentiated from the true pus secre- 

1 See Kiister, Kolle and Wassermann's Handbuch, 1913, VI, 458. 

- McConnell (N. Y. Med. Jour., 1916, CIV, 300) reports the finding of Vincent's fusi- 
form bacillus and spirillum in smears from the cervix. See Noguchi and Kaliski (Jour. 
Exper. Med., 1918, XXVIII, 559) for a study of the spirochetal flora of the normal female 
genitalia. 

^ See Curtis, Surg., Gyn. and Obs., 1914, XVIII, 299; Ibid., 1914, XIX, 25; Du Bois, 
Rev. Med. de la Suisse Rom., 1916, XXXVI, 133; Permar, x\m. Jour. Obs., 191 7, LXXV, 
652; Hymanson and Hertz, Ibid., 662. 



404 DIAGNOSTIC METHODS 

tions observed in the blennorrhagia of gonorrhea, as the former is not neces- 
sarily associated with pus formation. In pregnancy a slight catarrhal vagini- 
tis is not infrequent, so that leucorrhea is more apt to appear at such times. 
If the inflammatory process becomes intense, large shreds of epithelium may 
be found and ulceration followed by vaginovesical or vaginorectal fistulae 
may be observed. Such pathologic findings are usually the result of gonor- 
rhea. In slight catarrhal conditions of the vagina yellowish-gray patches 
may be seen, which are due to infection with mycotic fungi. 

It is not infrequent to find the trichomonas vaginalis in the vaginal prepa- 
rations. This organism has been previously discussed in the section on Feces^ 
to which the reader is referred. The oxyuris vermicularis as well as its ova 
have been reported in the vaginal discharge, but do not seem to have excited 
any pathologic changes. 

Purulent Secretions. 

True purulent secretions arising from the vagina are almost always due to 
the presence of the gonococcus.^ This organism is accountable for a large 
number of gynecological conditions, so that it is wise for the practitioner to be 
able to identify it both from its clinical manifestations as well as by its labora- 
tory detection. 

In doubtful cases cultures should be made and a portion of the pus 
dropped into the eye of a rabbit. The gonococcus itself may later be 
recovered from the conjunctival exudate. 

It is not to be assumed that the finding of the gonococcus in the vaginal 
discharge is necessarily evidence of a gonorrheal vaginitis or vulvovaginitis. 
It has been shown that the semen of the male as well as the urethral discharge 
may contain gonococci and that these may be introduced into a perfectly 
normal vagina without necessarily setting up gonorrhea. However, this is 
unusual. The gonococcus may arise from the urethra, the Bartholin glands, 
acute inflammatory processes of the uterus, or from a ruptured pyosalpinx. 
In any of these cases the gonococcus may be found in the vaginal discharge, 
so that the laboratory worker should be guarded in his diagnosis of a vaginitis. 
Further, suppurative processes which were originally due to the gonococcus 
may later take on a type of mixed infection or even become of the sterile 
type. This latter condition is especially observed in old Bartholinitis, metri- 
tis, and cystic salpingitis. The organisms usually associated with the gono- 
coccus in the mixed infection are the streptococcus, staphylococcus, colon 
bacillus and pseudodiphtheria bacillus. In chronic metritis or salpingitis it is 
not infrequent to find the tubercle bacillus as the causative agent. 

Fetid Secretions. 

In these conditions the pus is usually chocolate colored, has a fatty ap- 
pearance, is extremely repulsive, is frequently sanguinolent and contains 
numerous degenerated cells as a result of marked leucolysis. This condition 
is especially observed in puerperal infection and may be extremely severe. 

1 See Sharp, Jour. Infect. Dis., 1914, XV, 283; Trist and Kolmer, Arch. Pediat., 1Q15, 
XXXIT, 801. 



SECRETIONS OF THE GENITAL ORGANS 405 

(2) Uterine Secretions. 

Normally, the uterus has no secretion beyond a slight mucoid one which 
is recognizable clinically. In inflammatory conditions, during normal men- 
struation, or following abortion or parturition, certain types of discharges 
are observed which have some clinical importance. 

Menstruation. 
Under normal conditions the menstrual fluid is at first mucoid in char- 
acter, but within a short time red cells appear and later the discharge takes 
on almost the character of pure blood. This menstrual fluid should be bright 
red in color, should contain no clots and should be discharged without caus- 
ing active pain. This fluid contains red cells, leucocytes, and prismatic 
epithelial cells showing large areas of fatty degeneration. The duration of the 
menstrual period is variable, running between two and five days. The 
amount of blood lost under normal conditions averages about 200 grams, but 
may be much larger under pathologic conditions. Not infrequently do we 
find cases in which menstruation is associated with marked pain during more 
or less of the period of flow. This condition is known as dysmenorrhea and 
may be associated with the exfoliation of large shreds of mucous membrane, 
in some cases reported these shreds constituting almost a cast of the uterine 
cavity. To this latter condition is given the name membranous dysmenor- 
rhea. For the pathologic significance of these abnormal types of menstrua- 
tion as well as for a discussion of the condition associated with failure of 
menstruation (amenorrhea) the writer must refer to works on gynecology. 

The Lochia. 
By this term we have reference to the discharges from the uterine cavity 
during the puerperium. At first such discharges consist of blood, which may 
be in the form of clots, and decidual shreds along with epithelial cells which 
are probably of vaginal origin. This type is known as the lochia rubra or 
cruenta. During the next two or three days the discharges become paler and 
thinner, the red cells diminish and the leucocytes increase, while the decidual 
shreds may continue approximately the same. This type is known as the 
lochia serosa. After about a week the discharge assumes a grayish or yellow- 
ish color and a creamy consistency, the red cells diminishing rapidly and the 
white cells increasing markedly. Microscopic examination shows, besides 
the leucocytes and epithelial cells, numerous fat globules and cholesterin 
crystals. This discharge may continue during the remainder of the period 
of uterine involution and is known as the lochia alba. Under normal condi- 
tions the lochial discharge has a faint odor, but is never fetid. If a portion 
of the placenta or membranes have been retained, the lochia may assume a 
dirty brownish color and become extremely fetid. After the first two or three 
days numerous bacteria, such as staphylococci, streptococci and colon bacilli, 
may be present, but no untoward symptoms exist unless these, along with 
other saprophytes, give rise to a distinct puerperal infection or sapremia. 

Amniotic Fluid. 
This is a thin, whitish or pale-yellow fluid containing the constituents of 
ordinary transudates. The reaction is neutral or faintly alkaline, the specific 



4o6 



DIAGNOSTIC METHODS 



gravity varies between 1002 and 1008, and the amount of solids rarely reaches 
2 per cent. The albuminous bodies are principally vitellin, serum albumin, 
and traces of mucin, while glucose is absent. Urea and allantoin are present 
in traces, while creatinin has occasionally been reported. 

The amount of amniotic fluid varies between 700 and 1,000 c.c. Under 
pathologic conditions, however, this amount may be increased or decreased, 
giving rise on the one hand to polyhydramnios or dropsy of the amnion and 
on the other to oligohydramnios. For a discussion of these pathologic varia- 
tions as well as for a treatment of the subject of pathologic changes in the 
membranes, the writer will refer to works on obstetrics. 
Abortion. 
The recognition of abortion is usually made by examination of the material 
discharged from the uterine cavity. Usually one finds blood-clots in which 
the villi of the chorion are present. These usually appear as club-shaped 
masses with epithelial coverings, showing the characteristic capillary net- 
work. Moreover, decidual cells are usually present, and may be recognized 
by their large size, their round, polygonal, or spindle-shaped form, and their 
irregular and large nuclei with nucleoli.^ 



n 



^ 




Fig. 116. — Chorionic villi. {McMurrich.) 



Vesicular Mole. 

This condition has been called dropsy of the vilh of the chorion, hydatidi- 
form degeneration of the chorionic villi, cystic mole, and myxoma of the 
placenta. One of its most important symptoms is the expulsion through the 
vagina of the vesicles forming the degenerated mass. The mole is a mass of 
pedunculated vesicles which closely resemble a bunch of grapes or gooseberries. 
Each vesicle may vary in size from a millet seed to a large hazelnut and con- 
tains a fluid which is usually colorless and limpid, but may be reddish and 

1 See Lackner, Surg., Gyn. and Obs., 1915, XX, 537. 



SECRETIONS OF THE GENITAL ORGANS 407 

somewhat dense. Microscopic examination of the tissue shows the peculiar 
myxomatous degeneration of the chorionic villi. ^ 
Carcinoma. 
It is not infrequent to observe, in cases of severe hemorrhage through the 
vagina, the appearance of occasional shreds, which on microscopic examina- 
tion show the characteristic appearances of carcinoma of the cervix or body 
of the uterus. The diagnosis of carcinoma, however, would better be made 
upon sections removed by the surgeon rather than upon shreds found in the 
hemorrhagic fluids. Although a diagnosis may be at times possible, it should 
be somewhat guarded unless the clinical symptoms are distinctive. 

1 See Westermark, Hygiea, 1914, LXXVI, 801; Iraci, Policlinico, 1915, XXII, 1505; 
Amann, Monatsschr. f. Gebiirtsh. u. Gyn., 1916, XLIII, 11; Bar, Arch. Mens, d' Obs. et 
de Gyn., 1916, XXI, 49; Caturani, Amer. Jour. Obs., 191 7, LXXV, 591; Lopez, Med 
Ibera, 1919, IX, 5. 



CHAPTER VIII 

THE BLOOD 

I. General Considerations 

The blood is perhaps the most important tissue in the body, inasmuch 
as it is at once the purifier and the nutritive source of the cell. Any normal 
or abnormal product of cellular activity finds its way ultimately into the 
blood, either to be taken up by the assimilatory organs or to be thrown out 
by the excretory ones. While but relatively few disease processes are as- 
sociated with diagnostic findings in this tissue, yet many are characterized 
by definite manifestations which are invaluable aids to the clinician. 

It is, therefore, of the utmost importance that we should have a proper 
knowledge of the normal blood in order better to understand the various 
phases which characterize abnormal blood and which give to hematology 
such an interesting and important position in diagnosis. While it is true 
that some of the methods involved in hematological examinations require 
definite apparatus and a large experience for their proper interpretation, yet 
the results obtainable from the ordinary routine blood examinations are so 
invaluable, being in some cases pathognomonic, that no practitioner should 
consider himself fitted to give his patients the proper service without being 
equipped with a clear working knowledge of the methods of examination and 
the findings of normal and of abnormal blood. 

It is essential to remember that certain physiologic as well as pathologic 
conditions influence the quantity and quality of the blood. So great are the 
effects of digestion, exercise, nervous factors, massage, cold, heat, sweats, 
dysentery, constitutional and specific diseases, that one does not wonder at 
the many conflicting reports of cases showing widely varying hematological 
findings. As Grawitz has pointed out, no conclusion should be drawn from 
an examination of the blood without taking into consideration the physiologic 
and pathologic condition of the patient. 

It is a pleasure to observe in these days the tendency toward a more 
rational and thorough study of the plasma, the so-called "intracellular fluid" 
of the blood. We have forgotten, in our enthusiasm over the many valuable 
findings obtained from histological investigations, that the relations of the 
fluid portions of the blood are, in some cases, of quite as much importance as 
are the variations in the cellular elements. It is necessary only to cite the 
work on lysins, precipitins, agglutinins, opsonins, etc., to show the value of a 
more extended study of the plasma or serum. 

Regarding the technic of blood examinations, the writer will have much 
to say later, but he wishes to impress upon his readers one point which has 

408 



THE BLOOD 409 

been well expressed by Tiirk, namely, an indispensable basis for the proper 
utilization of any diagnostic, prognostic, or therapeutic method of cHnical 
examination is a knowledge of the absolute limitations of the method. Reli- 
able results can, however, be obtained only by those who are thoroughly 
familiar with the principles as well as with the technic and the little "knacks" 
of the method used. It will be found, when the attempts are made to apply 
the methods outlined, that quite as much depends on the exactitude with 
which the separate details are carried out as upon the selection of the method 
itself. It is not to be expected that a first trial will yield exact results or that 
a few determinations will perfect one in the methods of examination. Experi- 
ence is the only teacher that can equip one with the skill and power of inter- 
pretation necessary to cope with the many difficulties to be overcome in the 
hematological investigations. 

II. Physiology and Chemistry 

(i) Blood Formation and Blood-forming Organs. 

While it is impossible in a general work of this character to go into 
great detail regarding the formation of the blood, yet it seems to the writer 
that a brief discussion of this subject is extremely valuable both to the student 
and practitioner.^ 

Red Corpuscles. 

According to Kolliker, the first blood-corpuscles have their origin, in 
embryonal life, in the embryonic heart and blood-vessels. They appear 
as nucleated colorless cells, which later develop into colored corpuscles by 
the appearance of hemoglobin in some of the cells of the mesodermal cord, 
which cells go to form the first capillaries. Upon the formation of these 
vessels the cells lie within them as nucleated reds. At this time there are no 
true leucocytes and none appear until after the complete formation of the red 
cells, which is advanced as an argument against Pappenheim's theory 
of single origin of red and white cells. It will thus be noted that the vessel 
wall and the primitive erythrocyte have a common origin in the meso- 
dermal cord, the peripheral cells going to form the endothelium of the vessel 
and the internal cells the corpuscles. Up to the end of the fourth or fifth 
week of embryonal life all of the red cells are nucleated; while from that time 
on the relation of the nonnucleated to the nucleated forms gradually increases 
until at birth few if any nucleated cells obtain. 

In later embryonal life (about the third month), the liver becomes the 
chief seat of blood formation. During the fifth month, the spleen^ and lymph- 
glands take up this work, and finally the bone-marrow becomes the seat of 
such activity. 

In extrauterine life, the bone-marrow is the chief point of formation of 
the red cells, but under pathological conditions the spleen and liver may as- 
sume their embryonic functions. It appears that the formation of nucleated 

1 See Gruner, The Biology of the Blood Cells, New York, 19 14. 

2 See Morris, Jour. Exper. Med., 1914, XX, 379; Hirschfeld and Weinert, Berl, klin. 
Wchnschr., 1914, LI, 1026. 



41 DIAGNOSTIC METHODS 

reds in the adult is practically the same as in the embryo and that, at all 
periods of life, the red cell is the product of several series of mitoses of a 
colorless mesoblastic cell. The difficulty of tracing this series, from the 
large nucleated red cell to the colorless mesoblastic "mother-cell" in the 
marrow, has given rise to the diverse opinions now held regarding the ulti- 
mate development of the red corpuscles. 

Leucocytes. 

The earliest indications of the formation of leucocytes are seen in the 
presence of primary wandering cells, of mesodermal origin, which are found 
principally in the loose connective tissues of the early embryo. Though of 
mesodermal origin they are, from the first, quite distinct in morphology 
and, apparently, in function from the capillary endothelium and fixed con- 
nective tissue cells. Their development has been traced by Ziegler to masses 
of mesodermal cells surrounding the cords from which the capillaries are 
formed. It thus seems that originally the parent leucocytes lie outside the 
vessels, into which they make their way by virtue of ameboid powers. 

Most observers find that the primary wandering cells produce, by mitotic 
division, one or more generations of colorless cells which gradually approach, 
in morphology, the early basophilic leucocytes of the circulation. Denys, 
Lowit, Ziegler, von der Stricht, and others claim that red cells and white 
cells develop from separate series of cells, which have become differentiated 
from the primary mesodermal cells with the first appearance of blood and 
blood-vessels. Kostianecki, ^ Miiller, Schmidt, Saxer, Pappenheim, and 
others believe that the primary wandering cell persists, in the blood-forming 
organs as the parent of both red and white cells. 

Before the leucocytes begin to appear in the circulation, mitotic figures 
are abundantly seen in the primary wandering cells in various situations. 
These are gathered in groups, first in the loose connective tissues of various 
regions, where lymph nodes subsequently develop; but the chief seat of the 
production of the leucocytes is found in the embryonal liver. In both situa- 
tions the wandering cells are found in lymph and blood capillaries, in the 
interstices of the connective tissues, and between the liver cells. In later 
embryonal life the process is gradually transferred from the liver to the lymr 
phoid and adenoid tissues, as indicated by the development of lymph nodes, 
spleen, marrow, and thymus. Under normal conditions, the reproduction 
of leucocytes, in the adult, is limited to the lymphoid structures both of the 
lymph-glands and bone-marrow. 

(2) Total Volume of Blood. 

The various methods which have been advanced for the estimation of 
the total quantity of blood in the body are subject to such wide variations 
that they have yielded little exact information regarding this subject. The 
procedures advocated by Valentine, Vierordt, Buntzen, and Thibault have 
an error sufficiently great to exceed the physiological and pathological varia- 
tions of the blood. By these methods, the quantity of blood in the body 
has been estimated as equal to one-thirteenth of the body-weight. The so- 



THE BLOOD 41I 

called clinical methods of Quincke or of Tarchanoff are of purely theoretical 
interest, because certain factors, such as the appearance of the patient pi; 
the volume of the pulse, are taken into consideration in making a rather un+ 
reliable guess as to the total quantity of blood in the body (Buckmaster). 

By the use of a reliable method, introduced by Haldane and Smith,^ the 
total volume of blood may be fairly accurately estimated. This method is 
based on the following points. The capacity of hemoglobin for oxygen and 
for carbon monoxid is identical. On the assumption that none of this lattei; 
gas is oxidized in the body, and that no substance in the blood, other than 
hemoglobin, unites with it, the experimenter is able to determine the CQ 
capacity and hence the O capacity of the blood. This method has, however, 
little clinical appHcation and will be left with reference to the original work* 
In the cases studied by Haldane and Smith by. this method, the average 
value was 3,240 c.c. or, on the basis of a specific gravity of 1,060, about 3,434 
grams. Phis yields, according to Smith, a figure ranging between one-six- 
teenth and one- thirtieth of the body-weight. ^ In obese persons the volume 
of blood is less, proportionately, than in the more normal specimens of 
mankind. ; ,:.; 

The question of the volume of blood in the body is of great importance ,in 
the study of the changes taking place in this tissue. It must be remembered 
that the number of red or of white cells in a cmm. of blood will depend upon the 
total amount^! blood present. If for any reason the volume is diminished 
or increased, corresponding changes, in the inverse sense, will be observed 
in the number of the cellular elements per cmm. It seerns to the writer, there* 
fore, that certain factors, not ordinarily taken into account in blood examina- 
tions, should be known before any definite report is made upon a blood count. 
It may be readily seen that a concentration of the blood, due to hemorrhage, 
diarrhea, sweating, etc., will lead to an apparent increase in the number of 
corpuscles. 

Certain physiologic and pathologic conditions lead to definite changes 
in the volume of the blood, as such, or of some of its constituents. As Plehn 
has recently shown, the volume remains quite constant or is adjusted thrpugH 
the activity of the capillary endothelium and tlirough the influence of thi^ 
nervous system. However, definite changes of a more or less transitory nature 
do occur and exert marked influences on the results of blood examinations, 
as well as upon many pathologic conditions,^ . 

Oligemia. 

By this term is meant a reduction in. the total vplume;'of blood,^ both as 
regards the liquid and the celltdar portions. This condition is most f re- 

^ Jour. Physiol, 1896, XX, 497; Ibid., 1900, XXV^ 333, See Salvesen (Jour. Biol. 
Chem., 1919, XL, 109) for a new method of determining blood volume. His determina^ 
tions show the blood volume to average 1/16.8 of the body weight. ' J", 

2 Keith, Rowntree and Geraghty (Arch. Int. Med., 1915, XVI, 547) ronsider that the 
blood constitutes 8.8 per cent. (1/11.4) pf ' the body- weight. Normal individuals ha v^ 
approximately 85 c.c. of blood per kilograrn. ' ' 

2 See Kammerer and Waldmann, Deutsch. Arch. f. klin. Med:, 1913, CIX, 524; Miller^ 
Keith and Rowntree, Jour. Am. Med. Assn., 1915, LXV, 779.^ r , ' 



412 DIAGNOSTIC METHODS 

quently noticed after profuse hemorrhage^ and may be so marked that death 
results. In other cases, in which the hemorrhage is less extensive, the loss of 
blood is made up by osmosis from the lymph spaces into the capillaries and, 
later, by an increase of the cellular elements due to compensatory activity of 
the hematopoietic organs. 
Plethora. 

The opposite of the preceding condition is known as plethora, a state 
characterized by an increase in the total volume of blood. There has been 
much discussion as to whether a true plethora exists, but there can be little 
doubt that a transitory plethora vera may occur as a result of direct transfusion 
of blood, and also, according to Bergmann and Heissler, who have established 
the fact that there is a direct ratio between the volume of blood and the size 
of the heart, on the one hand, and the muscular development of the subject, 
on the other, as the result of increased muscular activity, provided the loss of 
fluid by perspiration is not excessive. Such a plethora disappears, of course, 
in a very short time. 

In this discussion we must distinguish between a serous and a cellular 
plethora. By the former is meant an increase in the volume of blood due 
to excessive quantities of its liquid and soluble cons titu tents; while by the 
latter we understand an increase in the number of corpuscular elements, that 
is a polycythemia. Serous plethora is most frequently observed in organic 
lesions of the kidneys and of the heart, in which a diminished elimination of 
water and inorganic constituents is noted. This condition is usually of a 
transient duration, as the volume of blood is soon restored to normal by intra- 
capillary transudation and by diffusion. Osmotic effects must also be taken 
into consideration here, inasmuch as the salts will tend to diffuse out from the 
blood and will consequently draw water after them, giving rise, under certain 
conditions, to dropsical effusions of a more or less transient duration. 
Hydremia. 

Another condition of the blood is frequently observed, in which an in- 
crease in the quantity of the liquid constituents is observed. This is known 
as hydremia and is different from serous plethora, as the latter carries with 
it an increase in the saline as well as in the watery portion of the blood. In 
hydremia the specific gravity of the blood is reduced, while in serous plethora 
it is increased. As EngeP has shown, the estimation of the coefl&cient of 
refraction of the blood serum is a reliable method for the clinical study of the 
subject of the water content of the blood. 

Hydremia may be produced by any factor which changes the normal 
relationship of the blood constituents in such a way that the watery portion 
is relatively increased. It is in these more or less physiologic states that we 

1 See Hogan, Jour. A. M. A., 1915, LXIV, 721; Bogert, Underbill and Mendel, Am. 
Jour. Physiol., 1916, XLI, 219; Scott, Jour. Physiol., 1916, L, 157; Guthrie, Jour. A. M. A., 
1917, LXIX, 1394; Arch. Int. Med., 1918, XXII, i; Penn. Med. Jour., 1918, XXII, 123; 
Meek and Gasser, Am. Jour. Physiol., 191 8, XL VII, 302; Robertson and Bock, Jour. Ex- 
per. Med., 1919, XXIX, 139; Aebly, Cor. Bl. f. Schweiz. Aerzte, 1919, XLIX, 1393; Lee, 
Am. Jour. Med. Sc, 1919, CLVIII, 570; Gasser, Erlanger and Meek, Am. Jour. Physiol., 
1919, L, 31 and 104. 

2 Magyar Orvosi Arch., 1906, VII, 555. 



THE BLOOD 413 

are apt to observe the greatest variation in the blood counts. As the cellular 
elements are not simultaneously increased, the drop of blood under examina- 
tion contains relatively fewer cells than normally. The most common 
physiologic causes of hydremia are the ingestion of large quantities of fluids, 
saline transfusions, and vasomotor dilatations as a result of exercise or nervous 
influences. In severe anemias we find the watery portion of the blood rel- 
atively increased. In dropsical states, following cardiac or renal lesions, we 
often observe such a condition, whose duration will depend, of course, upon 
the etiological factors of the trouble. 

Anhydremia. 

This is a condition characterized by a diminution in the liquid constituents 
of the blood. There is no change in the cellular elements and hence a blood 
count will show an erroneous increase in the number of cells. In this condi- 
tion the specific gravity of the blood is naturally increased. 

Anhydremia follows any condition which results in the excessive loss 
of fluid from the body, as, for instance, that following profuse diarrhea, poly- 
uria, sweating, vomiting, and effusions into the various serous cavities of the 
body. According to Oliver, this state may be caused by influences which in- 
crease the arterial tension and hence bring about an increase in the passage 
of water from the vessels into the tissues. Thus, for instance, we may observe 
anhydremia following local and general exercise, massage, bathing, etc. 

(3) Volume Relations of Cells of Plasma. 

The study of the relationship between the cellular and the intracellular 
portions of the blood is a comparatively recent addition to the technic 
of blood examinations. , This determination is based on the principle that the 
corpuscles may be thrown by centrifugal force to the distal end of a calibrated 
tube, while the plasma will collect in the proximal portion. If the tube be 
properly calibrated, the percentage relations of the cells and plasma may be 
readily ascertained. These ideas were used by Hedin in elaborating the 
earlier methods of Blix. 







Fig. 117. — Daland's hematocrit. 

Daland's Hematocrit. 

D aland has introduced a modification of the clumsy model of Hedin and 
has succeeded in simplifying the technic to such an extent that this method 
is directly applicable to clinical use. His instrument is shown in the accom- 
panying cut. 

One of the calibrated glass tubes is fitted with the rubber tubing and is 
filled with blood from the ear or finger. The forefinger, smeared with a 



414 DIAGNOSTIC METHODS 

little vaselin, is then placed over the beveled end of the tube and the rubber 
tubing withdrawn. Insert the tube into one arm of the frame, the other arm 
of which should carry the second tube filled in the same manner, in order to 
balance the instrument and to control the reading. Rotate the spindle for 
three minutes at such a rate of speed as will insure 10,000 revolutions per 
minute (80 revolutions of handle). In this way the corpuscles are separated 
from the plasma and are distinguishable as a distinct column, which may be 
i^ead off directly from the graduations of the tube. These divisions will give 
the percentage relations of the cells and plasma as the tube is divided into 
100 equal portions, each division of the scale representing approximately 
100,000 cells. This latter makes it possible to make a rather rough blood 
count with this instrument, but it is to be remembered that accurate results 
cannot follow, as we find such variations both in the size and elasticity of the 
cells in the different conditions in which the number of cells is most sought; 



Fig. 118. — Hematocrit tube. 

Volume Index. 

Capps^ has introduced the conception of volume index, that is the quotient 
of the volume per cent, as obtained with the hematocrit, and the blood count 
in terms of per cent. Sahli advises the use of the expression volume quotient 
or volume value for this factor. 

By means of the hematocrit the volume of the red cells, as compared with 
that of the whole blood, is taken. In normal cases this is about 50 per cent, 
which is reckoned as one. Hence the volume of the red cells may be obtained 
directly in percentage value. The red cells are then counted by the method 
to be later outlined, and the result is expressed in percentage by comparing this 
count with a so-called normal one of 5,000,000 red cells. By dividing the 
volume of red cells (in per cent.) by the per cent, of red cells, Capps obtains his 
volume index of the red cells. 

In normal conditions this quotient is one. According to Capps, an in- 
crease of this index is a constant factor in pernicious anemia. The color in- 
dex never exceeds the volume index in such cases, which fact shows that there 
is no supersaturation of the corpuscles with hemoglobin. In primary and also 
in secondary anemia this factor is diminished. Here we find the color index 
often falling below the volume index. 

This method may be used in detecting various pathological conditions of 
the blood. According to Emerson, it is used in the Johns Hopkins Hospital 
in ascertaining the presence of lipemia, cholemia, or hemoglobinemia. It 
would seem to the writer that it could be employed with advantage as a 
routine procedure, especially in hospital practice. The osmotic pressure of 
the plasma plays a great role in this determination, as the concentration of 
the blood may be such as to cause swelling or shrinkage of the cells. As 

^ Jour. Med. Research, 1903, X, 367. 



THE BLOOD 415 

Capps has found, normal red cells with a volume index of one have their dis- 
coplasm saturated with hemoglobin. Hence, if the hemoglobin index be- 
comes greater than one, an enlargement of the red cells is indicated. Upon 
the other hand, the color index may fall, regardless of a corresponding lower- 
ing of the volume index. It follows, therefore, that, if the color index is 
above normal, the volume index must also be increased; while, if the hemo- 
globin index is below normal, the volume index is not necessarily diminished 
(Sahli). 
(4) Methods of Obtaining Blood. 

The method of obtaining blood for examination depends upon the amount 
desired and on the examination which is to be made. For ordinary routine 
work only a few drops are necessary, while for bacteriological investigations 
2 to 20 c.c. may be required. 

In obtaining the blood no set rule can be laid down as to the proper place 
from which to take the specimen. We should select the part which promises 
the best results, avoiding naturally the points which are cyanosed, eczema- 
tous, edematous, hyperemic, cold, unduly heated, or, in other words, any part 
which is not normal. The ear usually furnishes the best results, in the 
writer's opinion, because its puncture is relatively painless, it is easily acces- 
sible (which point is often of importance in attempting to obtain blood from 
those who are comatose or who refuse to submit their hand for puncture), 
and because the patient, who may be easily affected by the sight of blood, 
can not see the drop. If the lobe of the ear is thick it is usually pricked on the 
flat side, but if it be thin it is well to make the puncture on the edge and par- 
allel to the surface. Some workers prefer the palmar surface of the ball of 
the middle or second finger of the left hand and others advise pricking the 
arm over a small superficial vein. In cases in which repeated examinations 
of the blood are to be made the parts should be varied in order to prevent 
soreness and also to avoid anesthesias which occasionally follow repeated use 
of the same site of puncture. 

Quite a number of special forms of blood needles, as, for instance, those 
of Francke and of D aland, are on the market, and each of them has its ad- 
vocates. Many of them are in the form of special holders, which permit a 
puncture of a desired depth to be made without any danger of going too deep. 
These are to be recommended to those only who seem unable to control their 
stab. The writer does not find that the results obtained by the student 
are any better with such instruments than are those following the use of the 
ordinary Hagedorn surgical needle. In lieu of any better article, a clean 
steel pen with one nib broken will yield admirable results. The one thing 
to bear in mind in selecting a needle for blood work is that the point have 
a cutting-edge and should not be round or sharp nor should it be too long 
or slender. 

Having decided upon the part from which the blood is to be taken, this 
surface is washed carefully with water and alcohol or ether and is then thor- 
oughly dried. Avoid any undue rubbing as this will cause hyperemia and 
will introduce an error into the work. It is, as a rule, unnecessary to sterilize 



4i6 



DIAGNOSTIC METHODS 



the needle but if it seems advisable to do so on account of the patient's attitude, 
the sterilization is best done by heat, hydrogen peroxid or alcohol, allowing 
the needle to cool before making the puncture. In this latter part of the 
technic, much depends on the amount of blood desired as regards the use of 
a short quick stab or a slow steady puncture. The latter procedure usually 
yields more blood but is more painful. It is much better to prick the patient 
too deeply, going even through the lobe of the ear, than it is to subject him to 
repeated punctures. The part pricked should not be squeezed nor held in a 
position which will cause an abnormal circulation. If the puncture is suc- 
cessful, the blood will come out in good-sized drops. The first of these are 
wiped away and subsequent ones used for the examination. As often hap- 
pens, the blood coagulates fairly quickly, so that the coagulum should be 
wiped off with a little alcohol followed by a dry cloth. It is important to 
remember that a patient with hemophilic tendencies or history may bleed 
very easily from a very slight puncture. Care should, therefore, 
be taken to question the patient regarding the ease with which 
blood flows from a wound and also regarding the history of 
' ' bleeders ' ' in the family. 

If considerable blood is desired, resort must be made to venous 
puncture. The site of this operation is usually the median basilic 
vein at the bend of the elbow. This vein may be made more 
prominent by tying a tight bandage around the arm, but the band- 
age should be removed before the blood is withdrawn, except when 
serum reactions are to be studied. The site of puncture must be 
g thoroughly cleansed, using the precautions observed before any 
^^ surgical operation. Likewise, the needle and the aspirating 
instrument must be absolutely sterile before puncture is made. 
The instrument best adapted for this purpose is, in the writer's 
experience, the Liier syringe, which is made of glass with a tightly 
fitting glass plunger and adjustable platinum needle. From 2 to 
20 c.c. of blood are withdrawn and immediately placed in work. 
The question of the bacteriological examination and of serum 
reactions will be discussed in a later section. 

(5) Physical Properties. 

The blood must be regarded as a fluid tissue, consisting of a 
transparent liquid, the plasma or liquor sanguinis, in which are 
suspended the corpuscular elements, erythrocytes, and leuco- 
cytes. Besides these latter cells we find two constituents, the 
blood plates of Bizzozero and the hemoconien (blood dust), which 
'are hardly to be considered as true corpuscular entities. 
As it flows from the vessels, blood is a thick, viscid, red liquid, having 
a peculiar odor, a salty taste, and an alkaline reaction to litmus. If allowed 
to stand it shows, unless certain precautions are observed, the peculiar 
phenomenon of coagulation. In this process the blood is separated into two 
portions, the cellular elements and the plasma, the latter changing, as the 



Fig. 119. 
Blood needle. 



THE BLOOD 



417 



/ 



process goes on, into serum and the clot (fibrin), which holds the corpuscles 
in its meshes. In the diagram given below, adapted by Webster and Koch, 
may be seen the composition of the blood. 

Serum Albumin. 



Plasma 



Blood 




Serum 




Fibrinogen - 



Serum Globulin. 

Glucose, extractives, calcium salts, sodium 
and potassium chlorids, carbonates, phos- 
phates, tte. 
(yields fibrin). 



Cellular Elements 




Red Corpuscles 

White Corpuscles' 
Blood Plates. 




Oxy-hemoglobin. 

Lecithin. 

Salts. 

■Fibrin Ferment. 



Hemoconien. 'B^ 



o\ 



(A) Color. 

The color of the blood is due to the presence in the erythrocytes of an iron- 
containing albuminous substance, hemoglobin, which has remarkable affinity 
for oxygen and other gases. This latter property enables this pigment to 
play one of the most important roles in the body economy. Arterial blood 
is bright red in color, while venous blood shows a purplish-blue tint. These 
variations are due entirely to the relative proportions of oxygen and of carbon 
dioxid in the two types of blood. Many different shadings are observed, 
physiologically and pathologically, in the coloration of the blood, and each is 
due to some combination of hemoglobin with normal or abnormal substances. 

The presence of large numbers of red corpuscles in the blood gives rise to a 
characteristic opacity of this tissue. If, for any reason, such as admixture 
of blood with water, dilute salt solutions, urea, ether, snake venom, extract 
of mushrooms, etc., the blood loses its opacity, the change is due to the dis- 
solving out of the hemoglobin from the red" cells. This is the well-known 
phenomenon of "laking" or, better, of hemolysis, about which we will have 
something to say later. ^ 

The normal color of the blood is often changed in pathological conditions. 

Thus, in anemia the blood is pale and watery; in leukemia it may be milky; 

in diabetes buff-colored; while in poisoning with potassium chlorate it is 

chocolate-colored and in that with carbon monoxid it is bright red.^ 

^ See Guthrie, Jour. Lab. and Clin. Med., 191 7, III, 87. 
2 See Gaisbock, Med., Klinik, 191 2, VIII, 1906. 



41 8 DIAGNOSTIC METHODS 

{B) Odor. 

The odor of the blood is pecuHar and indescribable. This halitus san- 
guinis is due to the presence of certain volatile fatty acids and may be ren- 
dered more distinct by the addition of concentrated sulphuric acid, which 
increases the volatility of these acids (Barruel's test). 

(C) Reaction. 

If we are to accept the teachings of physical chemistry, that the alkalinity of 
a solution is due to the presence of free hydroxyl (OH) ions and that its acidity 
depends on the surplus of free hydrogen (H) ions, we must grant that the 
blood is a practically neutral fluid. If, however, we have in mind the acid- 
combining power of the blood, we must regard the reaction of this tissue as 
alkaline. It is certain that the blood shows both acid and alkali combining 
powers due to the presence of protein constituents as well as to both acidr 
and alkali-reacting substances, the measure of such powers being dependent 
on the indicator used in the estimation. As the combining power for acids is 
greater, in the case of blood, than it is for alkalies, the reaction, as judged by 
titrimetric methods, must be alkaline. 

The normal free or diffusible alkalinity of the blood is due to the presence 
of disodium hydrogen phosphate (Na2HP04), sodium bicarbonate (NaHCOs), 
and sodium carbonate (Na2C03). This total diffusible alkali constitutes, 
according to Brandenburg, about 20 per cerit of the entire alkalinity and may 
be measured by dialyzing against known alkaline solutions and observing the 
concentration at which the strength of the known solution does not change. 
This factor represents the so-called alkaline tension of the blood and remains 
fairly constant, in normal cases, at about 60 mg. of NaOH per 100 c.c. of blood, 
while in pathologic conditions, such as uremia, diabetes, etc., it is somewhat 
reduced. 

Besides this diffusible alkali, the blood contains nondiffusible alkali bound 
to the proteins. This portion represents normally about 80 per cent, of the 
total alkalinity, and is dependent largely on the cellular content of the blood, 
as the soluble protein constituents are not generally subject to wide varia- 
tions. The fluctuations in this nondiffusible alkali are no doubt accountable 
for the great differences in the figures given for the* alkalinity of the blood. , 

The subject of the reaction of the blood is one which. should furnish, if 
properly studied by reliable methods, much valuable data upon subjects 
which are now very obscure. As Moore and Wilson^ have pointed out, 
the titration methods do not give us the true neutrality of the blood, but 
rather the amount of alkali or of acid which may be added to it without rais- 
ing the hydroxyl or hydrogen concentration above certain low limiting values. 
The reaction of the blood depends on the indicator used for the determina- 
tion of the neutral point and cannot be definitely measured by any method 
which employs an indicator for such purposes. These writers have intro- 
duced the term ''reactivity" to indicate the property, possessed by the 

^ Biochem. Jour., 1906, I, 297. See, however, Sellards, Bull. Johns Hopkins Hosp., 
1914, XXV, loi. 



THE BLOOD 419 

blood, of combining with both alkalies and acids in such a way as not to raise 
its ionic composition. 

In discussing the methods, which are now in use, for the determina- 
tion of the reaction of the blood, one must have in mind just what is meant 
by the terms "acid" and "bases," from the physico-chemical point of view.; 
When the molecule of hydrochloric acid, for instance, is dissolved in water it 
splits or dissociates into two portions called ions, H and CI, each bearing an 
electric charge of opposite character. The electric charge of H, as well as of 
all metals, is positive, while that of CI, as of all acid groups, is negative.: 
As unlike charges of electricity are attracted by each other, it follows that: 
the H ions will collect at the negative pole, or cathode, while the CI ions 
will pass to the positive pole, or anode. It is for this reason that positive' 
ions are called "cations" and negative ions "anions." The more modern 
definition of an acid is a solution which contains an excess of hydrogen j 
over hydroxyl ions; neutrality is indicated by an equal number of hydrogen, 
and hydroxyl ions; while basicity (alkalinity) is denoted by an excess of 
hydroxyl ions over hydrogen ions. In the combination of acids with bases 
the hydrogen ions unite with the hydroxyl ions forming water, and thus re- 
moves the free ions from the field. It is evident, therefore, that the most 
strictly neutral solution must be pure water, in which the H and OH ions are 
exactly balanced. It has been shown that, even in the purest water, a slight 
degree of dissociation occurs, giving us free H and OH ions. The equation, 
used in physical chemistry, to indicate the dissociation of water is the follow- 
ing, which is reversible; H20^HH-0H. From the above considerations, 
it is evident that there must be an equilibrium between the H and OH ions, 
which is expressed in the equation, (H)X(OH) = Kw, that is the product of 
the free H ions by the free OH ions is a constant, which is called the "dissocia- 
tion constant of water." When the concentration of these ions is mul-. 
tiplied, in the purest water, the product is 1.2 Xio"^"^, which means that therC) 
are 1.2 gram molecules of hydrogen and hydroxyl present in 10,000,000,000,- 
000 liters. As H and OH are to be considered equal, the H ions must be 1.2 
Xio~", which signifies that this is present in sufficient quantity to form a 
0.000,000,12 N solution, or in other words, 1.2 grams of H in 10,000,000 
liters. In the study of solutions, the standard of concentration is taken as 
the "Normal" solution, which, by definition, contains the equivalent of i 
gram of hydrogen in i liter of the solution. 

If the dissociation constant of water, as mentioned above, is to remain a 
constant, there must be present in every aqueous solution some H ions and 
there must be maintained a balance between the concentrations of H and OH. 
For this reason, when acid is added to water the concentration of H ions 
necessarily rises, but the concentration of OH ions falls correspondingly, so 
that, as in pure water, HX0H = Kt^=i.2Xio~^^ No matter how acid or 
alkaline a solution may be, the products of these concentrations must be 
the same. It follows, therefore, that solutions with H ion concentrations- 
greater or OH ion concentrations less than 1.2X10"^ are called acid solutions;^ 
while those with H ion concentrations less than or OH ion concentrations^ 



420 DIAGNOSTIC METHODS 

greater than the above are called alkaline solutions. It will thus be seen 
that acidity and alkalinity may both be expressed in terms of H ion concen- 
tration. In order to avoid writing large figures for the acidity or alkalinity 
in expressing the normality of a solution, the logarithmic notation is used, 
the H ion concentration (Ch) being expressed in negative coefficients of lo. 
Thus, a i/ic, 000,000 N solution would be expressed as 10""'^. It has, how- 
ever, become customary to use the notation suggested by Sorensen, for a 
matter of convenience, and designate the H ion concentration by Ph, which 
is the logarithm of the reciprocal of the H ion concentration. Thus the H 
ion concentration of a 1/10,000,000 N solution would be written, Ph'. ■ As 
both notations are used in the literature, it is necessary to know how to con- 
vert them for purposes of comparison. A convenient method is to find the 
logarithm of the number of gram molecules of H and subtract this from the 
characteristic. Thus to convert the expression 1.2X10"^ into terms of 
Ph, the logarithm of 1.2 is 0.060206; hence 7 — 0.0602 = 6.94. The follow- 
ing table will serve to indicate these relations: 

Ph 

5-0 
5-1 
5-2 
5.3 

5-4 
5-5 
5.6 
5.7 
5.8 

5-9 
6.0 
6.1 
6.2 

As Ph increases, Ch decreases, and vice versa. Hence, values lower than 7 
denote increase of acidity and values greater than 7 denote lessened acidity 
or alkalinity. In the case of solutions of varying normality, this may be 
seen from the following table 

Phi indicates N/io acid 

Ph2 indicates N/ioo acid 

Ph3 indicates N/iooo acid . • 

Ph* indicates N/ioooo acid 

Ph5 indicates N/iooooo acid 

Ph« indicates N/i 000000 acid 

Ph7 indicates N/iooooooo acid (Neutrality) 

Ph8 indicates N/i 000000 alkali 

Ph9 indicates N/iooooo alkali 

Ph'o indicates N/ioooo alkali 

Phu indicates N/iooo alkali 

Ph12 indicates N/ioo alkali 

Ph'3 indicates N/io alkali 

On the addition of an acid or an alkali to water or normal salt solution 
a change in Ph must occur. However, when acid or alkali is added to whole 
blood, plasma, or serum, the H ion concentration does not change to any 
appreciable extent until quite an excess has been added. It is for this 
reason that the ordinary titrimetric methods for determining the alkalinity 
of blood are of Uttle avail. This is simply another way of expressing the fact 





Ch 




I 


oX 


io-» 


7 


9X 


io-« 


6 


3X 


io-« 


5 


oX 


io-« 


4 


X 


ic^e 


3 


2 X 


10-6 


2 


5X 


10-6 


2 


X 


10-6 


I 


6X 


10-6 


I 


2 X 


i<^6 


I 


oX 


10-6 


7 


.9X 


10-^ 


6 


3X 


10-' 



THE BLOOD 42 I 

that in health and in disease the reaction of the blood does not vary much 
from the usual value of Ph^, the factor being a physiologic constant. Even 
the slightest increase in Ch is to be regarded as incompatible with life if 
continued for any length of time. The power of certain solutions, such as 
blood, to resist change in reaction, on addition of acids or alkali, is due to 
what is styled the "buffer" or "tampon" action of these solutions. In the 
case of whole blood, the presence of both alkaline and acid phosphates, of 
carbonates and bicarbonates, and of proteins, which serve as both weak 
acids or bases, affords the maximum opportunity for the display of this buffer 
action. In so far as the plasma is concerned, this buffer action is principally 
due to the presence of bicarbonates. Through the work of several investi- 
gators, especially Henderson and his co-workers and Van Slyke and his col- 
leagues, the influence of these substances has been very carefully studied 
and methods evolved for the determination of the bicarbonates of the blood 
and their importance as indicators of varying degrees of acidosis. It has 
been found that the hydrogen ion concentration of the blood is directly pre- 
portional to the ratio existing between CO2 in solution as H2CO3 and sodium 
bicarbonate (NaHCOs) multiplied by a constant. This ma}^ be expressed as 

Ph = the molecular ratio -.t TTr^r^ • This ratio is normally — . 

NaHCOs ^ 2a 

It is evident, therefore, that while Ph is maintained with great tenacity by 
the normal organism, that the bicarbonate value decreases progressively as- 
soon as the normal excess of bases over acids begins to be noted. As will be 
seen later the organism, through the respiratory mechanism, rids the body of 
the excess of CO2 formed by the action of acids upon the bicarbonates and 
thus maintains the normal ratio between the H2CO3 and NaHCOs, and, in 
consequence, the Pn is kept normal until a large part of the bicarbonate has 
been exhausted. Hence variation in the Ph value is noted as a late change, 
while the value for the bicarbonate of the plasma changes from the beginning 
of the introduction of acid. Thus, if acid be added to the blood, owing to 
increased production in pathological processes or by introduction from with- 
out, it would react with the NaHCOa to form a neutral salt and, consequently, 
set free some H2CO3. The effect would be a diminution of the denominator 
of the normal ratio of i : 20 with a resultant increase of this value. The in- 
crease in Ph would not be proportional to that of the added acid, on account 
of the buffer action but it would nevertheless tend to show a slight change. 
If this acid were being added continuously, the hydrogen ion concentration, 
would rise unless some method existed for neutralizing the effect by decreas- 
ing the numerator at the same time that the denominator was decreased. 
Whenever, either by increased rate of CO2 production or by decomposition 
of NaHCOs by acid, the normal ratio of i : 20 is increased, the hydrogen ion 
concentration is proportionally increased. Whether it is the increase in 
hydrogen ion concentration or the accumulation of CO2 that is effective, the 
fact remains that the respiratory center is stimulated so that more rapid 
ventilation of the lungs follows until the H2CO3 is so reduced that the normal 



42 2 DIAGNOSTIC METHODS 

ratio of i : 20 is restored. The work of Scott would seem to indicate that CO2 
itself may excite the center quite independently of the H ion concentration 
of the blood. Boothby reports that the heart output is increased in the 
effort to rid the body of the excess of CO2. To distinguish the stage of 
acidosis in which the respiratory mechanism no longer keeps the CO2 concen- 
tration of the blood down to the normal value of 1/20 of the bicarbonate and 
in which the Ph does actually increase, Hasselbalch and Gammeltoft use the 
term ''uncompensated acidosis;" so long as the respiration, in spite of a 
diminution in the amount of bicarbonate, is able to keep the relations down 
to normal limits, the condition is known as ''compensated acidosis." We 
may, therefore, define acidosis as any condition in which the proportion 
between H2CO3 and NaHCO^ becomes greater than i: 20; or as Van Slyke 
defines it, a condition in which the concentration of bicarbonates in the 
blood is reduced below the normal level. Since the pulmonary epithelium 
permits the free CO2 of the blood to diffuse readily through it, it follows 
that the percentage of CO2 in the alveolar air must be a measure of the 
available NaHCOs in the blood. As MacLeod expressed it, "To repeat, for 
this is the fundamental conception of the whole acidosis problem, since Ch 
remains constant in the blood, the ratio H2CO3 : NaHCOs must also remain 
at its normal value of 1/20, and, therefore, if NaHCOs declines, H2CO3 must 
decline proportionately, and since this diffuses as CO2 into the alveolar air, 
the percentage of this gas in the latter must be proportional to the degree to 
which foreign acid can be added to the blood without perceptibly changing 
Chj in other words it must be proportional to the reserve alkalinity. One 
Other factor must clearly come into play to permit of the smooth operation 
6f the above mechanism, namely, the ratio of pulmonary ventilation must be 
adapted to' the amount of CO2 that has to be eliminated. This adaptation 
depends on the respiratory center, the activity of which is preeminently de- 
pendent upon the acid base equihbrium in the blood." As the free CO2 is 
eliminated the bicarbonate of the blood decomposes, because of the presence 
of other acid groups in the blood, and the amount that is left indicates the 
remaining ability of the blood to withstand further addition of foreign 
acid. "Clearly, therefore, the important thing to measure, in order that we 
may be enabled to diagnose the incipient stages of acidosis, is the alkaline 
reserve." 

The methods for the determination of the degree of acidosis have been 
many, applied both to the blood and urine. I select, therefore, those that 
seem to offer the best criteria from which to judge of this condition of acidosis. 

i. Van Slyke and Cullen's Method for CO2 Combining Power of 

Plasma 

This determination is made by the aid of Van Slyke's apparatus. It 
consists essentially of a 50 c.c. pipet with three-way cocks at top and bottom, 
and a i c.c. scale on the upper stem, divided into 0.02 c.c. divisions. At 
the bottom the apparatus is connected by a heavy walled rubber tube with 



THE BLOOD 423 

a levelling bulb filled with mercury. The right hand chamber below the 
lower stop cock serves to draw off the solutions, after the CO2 has been 
extracted from them, the other bottom (left hand) connection serving for 
subsequent release of the vacuum by the entrance of mercury. The appa- 
ratus is made of strong glass, in order to stand the weight of mercury without 
danger of breaking, and is held in a strong screw clamp, the jaws of which are 
lined with thick pads of rubber. The curved capillary above the upper stop 
cock is used for removal of solutions from the apparatus. This may, also, 
be used, for special gas analyses, to connect the apparatus with an absorption 
pipet. Three hooks or rings are attached to the stand holding the apparatus 
in such a position that the leveling bulb may be placed in one or other of 
these positions, as occasion may require. For purposes of the later descrip- 
tion of the technic, these positions are known as i, 2 and 3; the position i being 
such that the lower end of the levelling bulb is even with the upper stop 
cock of the apparatus; position 2 is such that the lower end of the levelling 
bulb is even with the lower end of the pipet itself below the lower stop 
cock; and the position 3 is 80 cm. below position 2. The calibrated upper 
stem of the pipet is of such diameter that i mm. of length corresponds to 
about o.oi c.c. By estimating tenths of a 0.02 c.c. division, gas volumes may 
be read to 0.002 c.c. In order to justify such readings, the apparatus must 
be accurately calibrated. It is essential that the stop cocks, especially the 
lower one, shall be held in place so that they can not be forced out by pressure 
of the mercury. For this purpose rubber bands may be used, but it is more 
advisable to employ elastic cords of fine wire spirals, as these are stronger 
and more durable. 

Before a determination is made, the entire apparatus, including the 
capillaries above the upper stop cock, is filled with mercury, a small bottle 
being placed under the curved capillary to catch any excess of the mercury 
forced out. To test the apparatus for tightness and freedom from gases, 
the mercury levelling bulb is lowered to position 3, so that a Torricellian 
vacuum is obtained, the mercury falling to about the middle of the right 
hand chamber below the lower stop cock. The levelling bulb is then raised 
again. If the apparatus is tight and gas-free the mercury will refill it com- 
pletely and strike the upper cock with a sharp click. In case there is any 
gas in the apparatus, this serves as a cushion so that the click is not heard 
and a bubble remains above the mercury. If this is the case, the apparatus 
must be repeatedly evacuated until the gas has all been removed. 

Collection of Specimen for Examination. — For at least an hour before the 
blood is drawn, the subject should avoid vigorous muscular exertion to pre- 
vent any lowering of the bicarbonates of the blood through the production 
of[lactic acid. The blood is drawn from the median basilic vein into a cen- 
trifuge tube containing enough powdered potassium oxalate to make about 
0.5 per cent, of the weight of the blood drawn. In this process it is advisable 
to^avoid stasis, or when stasis is necessary, to release the ligature as soon as 
the vein is entered and allow a few seconds for the stagnant blood to pass. 
While Van Slyke and CuUen state that results of similar significance are 



424 DIAGNOSTIC METHODS 

obtained by analysis of either whole blood or plasma, yet, as Macleod points 
out, there can be little doubt that the whole blood should be used as a part 
of the alkaline reserve is resident in the corpuscles. However it seems wise 
to employ plasma and sacrifice a certain degree of accuracy in the method, 
as this is easier to handle and measure and, in addition, keeps for a longer 
time without alteration than does whole blood. After collecting the blood, 
as above, this is centrifuged and the plasma drawn off with a pipet. Place 
3 c.c. in a 300 c.c. separatory funnel. To avoid possibility of error while the 
sample is awaiting analysis, the plasma is now saturated with CO2 at a defin- 
ite tension, namely, that of alveolar air containing approximately 5.5 per 
cent, of CO2. The funnel containing the plasma is turned on its side and the 
air within is displaced by either alveolar air from the lungs of the operator or 
with 5.5 per cent. C02-air mixture trom a tank. In either case, the gas mix- 
ture is passed through a bottle containing glass beads, to cool the air and 
condense the moisture of the breath. When alveolar air is used, as is most 
common, the operator, without inspiring more deeply than normal, expires as 
quickly and as completely as possible through the bottle of glass beads and 
the connecting separatory funnel, the stopper of the funnel being inserted 
just before the expiration is finished. In order to saturate the plasma with 
the CO2, the stoppered tunnel is turned end over end for 2 minutes, the plas- 
ma being distributed in a thin layer as completely over the surface of the 
interior as is possible. The funnel is now placed upright and allowed to stand 
a few minutes until the fluid has gathered in the bottom of the funnel. 

Determination. — The apparatus, including both capillaries above the 
upper cock, is entirely filled with mercury, and the cup at the top washed free 
of acid with carbonate-free ammonia, of about i per cent, concentration. 
Not more than 3 or 4 drops of this ammonia are necessary. The i c.c. of 
plasma, necessary for the analysis, is drawn from the funnel by means of an 
an Ostwald pipet and placed in the cup of the apparatus, the tip of the pipet 
dipping below the solution in the cup. With the mercury bulb at position 

2, as mentioned above, and the lower cock in such a position that a connection 
is established between the pipet and the right hand chamber below the cock, 
the solution in the cup is admitted to the pipet by opening the upper cock, 
leaving just enough solution above the upper cock to fill the capillary of the 
cup. The cap is now washed twice into the pipet with about 0.5 c.c. of water 
each time, a very small drop of caprylic alcohol (to prevent foaming) is added, 
and finally 0.5 c.c. of 5 per cent, sulphuric acid is run in. The total volume 
of the water solution run in must extend exactly to the 2.5 c.c. mark on the 
apparatus. After the acid has been admitted a drop of mercury is placed in 
the capillary and allowed to run as far as the cock in order to seal it. What- 
ever excess of the sulphuric acid remains in the cup is washed out with a little 
water. After all the solutions are in the pipet, the upper cock being closed 
and sealed with mercury, the mercury bulb is lowered and hung at position 

3, and the mercury in the pipet is allowed to run down to the 50 c.c. mark, 
producing a Torricellian vacuum in the apparatus. When the mercury 
(not the water) meniscus has fallen to the 50 c.c. mark, the lower cock is 



THE BLOOD 425 

closed and the pipet is removed from the clamp. Equilibrium of the CO2 
between the 2.5 c.c. of water solution and the 47.5 c.c. of free space in the 
apparatus is obtained by turning the pipet upside down 15 or more times, 
after which the pipet is replaced in the clamp. By turning the lower cock, 
the water solution is now allowed to flow from the pipet completely into the 
right hand lower chamber without, however, allowing any of the gas to 
follow it. The levelling bulb is then raised in the left hand, while with the 
right the cock is turned so as to connect the pipet with the lower left hand 
chamber. The mercury flowing in from this chamber fills the body of the 
pipet and as much of the calibrated stem at the top as is not occupied by 
the gas extracted from the solution. A few hundredths of a c.c. of water, 
which could not be drained off, float on top of the mercury, but this may be 
disregarded as far as the possibility of reabsorption of CO2 is concerned. 
The levelhng bulb is then placed at such a level that the gas in the pipet is 
under atmospheric pressure, that is the surface of the mercury in the levelling 
bulb should be raised until it is level with the mercury meniscus in the 
pipet, and, for entire accuracy, raised above the latter meniscus by a dis- 
tance equal to 1/14 the height of the column of water above the mercury 
(this latter precaution introduces, however, such a small error, that it may 
be ordinarily neglected). The volume of the gas is now read on the scale. 

Calculation.— WhQn from plasma, saturated as above described with al- 
veolar air, gases are extracted for analysis, one obtains not only the CO2 
bound as bicarbonate and set free by acidification, but, also, the CO2 and air 
physically dissolved by the plasma and water. These amounts may, of 
course, be determined by blank analyses or calculated from the known solu- 
bility coefficients of the gases. By taking into consideration all corrections 
necessary. Van Slyke and Cullen have formulated an equation, which permits 
the determination of the CO2 bound as bicarbonate by the plasma. This 
equation is as follows: 

T> 

X = -— (100.8 — o.27t)(V — o.i36+o.oo2t) 

in which B equals the observed barometric pressure; t equals the observed 
temperature at which the analysis and readings were made; V represents the 
actual reading, in mm., of the gas in the pipet; X expresses the c.c. of CO2 
reduced to 0° temperature and 760 mm. pressure, which i c.c. of plasma will 
bind as bicarbonate when in equilibrium at 20° with air containing 5.5 per 
cent, by volume of CO2. Van Slyke and Cullen have prepared a table from 
which the amount of CO2 bound as bicarbonate by ico c.c. of plasma may be 
read, at a glance, in terms of amount of gas shown in the pipet of the appa- 
ratus. 

By this method, the average normal value for man is found to be 65 
volumes per cent, of CO2. Austin and Jonas give as the minimum normal 
figure 60 instead of the 53 which Van Slyke and Cullen stated. From the 
preceding discussion, it is evident that this value must fall in all conditions 
associated with an acidosis, so that we find quite low values in diabetes. 



426 DIAGNOSTIC METHODS 

marked nephritis, most infectious diseases, etc. In severe secondary anemia 
and in pernicious anemia these values may be low, while in chlorosis the 
normal values usuallv obtain.^ 



2. Van Slyke, Stillman, and Cullen's Titration Method 

As the above workers state "In order to titrate accurately the bicarbonate 
of the blood plasma, it is both theoretically and actually necessary, as a 
net result of the operation, to transform the bicarbonate into the salt of 
whatever acid is used in the titration, without altering the normal hydrogen 
ion concentration of the plasma. If the hydrogen ion concentration is al- 
tered, the proteins of the plasma bind as a result either acid or alkali in 
amounts different from those which they bind in vive. Consequently, 
under such conditions the proteins act either to increase the acid added in 
titration to an amount more than equivalent to the bicarbonate, or to de- 
press it below the bicarbonate equivalent." The three requirements of a 
method, which will permit of the interpretation of the results in terms of 
plasma bicarbonate are (i) the use of the hydrogen ion concentration of 
normal blood as the end point; (2) removal of the CO2 set free by the added 
add, and (3) use of an indicator not affected by the plasma proteins. These 
have been combined in the following method. 

In drawing and centrifuging the blood the precautions outlined above in 
the discussion of the CO2 Capacity Method, for preventing loss or accumula- 
tion of CO2 and consequent change in the distribution of bicarbonate between 
corpuscles and plasma, are to be observed. Up to the beginning of the 
analysis, the blood and plasma are handled exactly as described in the pre- 
vious method. 

Technic. 

Two c.c. of the oxalated plasma, saturated with CO2, are pipetted into a 
round-bottomed flask of 150 to 200 c.c. capcity, and 5 c.c. of 0.02 N hydro- 
chloric acid are added (this is about 2 c.c. of acid in excess of the bicarbonate 

^ Van Slyke and CuUen, Jour. Biol. Chem., 1917, XXX, 289; Van Slyke, Ibid., 347. 
See, in this connection, Lundsgaard, Biochem. Ztschr., 191 2, XLI, 247; Henderson, Arch. 
Int. Med., 1913, XII, 153; Christiansen, Douglas, and Haldane, Jour. Physiol., 1914, 
XL VIII, 246; Marriott, Jour. Biol. Chem., 1914, XVIII, 507; Hopkins, Lancet, 1914, I, 
1589 and 1616; Boothby and Peabody, Arch. Int. Med., 1914, XIII, 497; Fridericia, Berl. 
kUn. Wchnschr., 1914, LI, 1268; Lewis and Barcroft, Quart. Jour. Med., 1915, VIII, 97; 
Van Slyke, Stillman, and CuUen, Proc. Soc. Exper. Biol, and Med., 1915, XII, 165 and 184; 
Menten and Crile, Am. Jour. Physiol., 1915, XXXVIII, 225; Peabody, Arch. Int. Med., 

1915, XVI, 955; Am. Jour. Med. Sc, 1916, CLI, 184; Marriott, Arch. Int. Med., 1916, 
XVII, 840; Walker and Frothingham, Ibid., XVIII, 304; Frothingham, Ibid., 717; Macleod, 
Jour. Lab. and Clin. Med., 1916, II, 54; Hornor, Boston Med. and Surg. Jour., 1916, LXXV, 
148; Austin and Jonas, Am. Jour. Med. Sc, 1917, CLIII, 81; de Almeida, Brazil-Medico, 

1916, XXX, 203; Van Slyke, Stillman, and CuUen, Jour. Biol. Chem., 1917, XXX, 401; 
Stillman, Van Slyke, Cullen, and Fitz, Ibid., 405; Henderson and Morriss, Ibid., XXXI, 
217; McClendon, Shedlov, and Thomson, Ibid., 519; Henderson, Ibid., XXXII,' 325; 
Palmer and Van Slyke, Ibid., 499; Friedman and Jackson, Arch. Int. Med., 1917, XIX, 
767; Whitney, Boston Med. and Surg. Jour., 1917, CLXXVII, 225; Arch. Int. Med., 1917, 
XX, 931; Pearce, Am. Jour. Physiol., 1918, XLV, 550; Scott, Ibid., XL VII, 43; Henderson 
and Haggard, Jour. Biol. Chem., 1918, XXXIII, 333; Macleod, Jour. Lab. and Clin. Med., 
1919, IV, 315. , 



THE BLOOD ' 427 

normally present, as this ranges between 0.03 and o.oi M). In order to re- 
move the CO2 set free b}^ the acid, the flask is shaken vigorously with a rotary 
motion for i minute, so that the solution is whirled in a thin layer about the 
inner wall of the flask. The solution is now poured as completely as possible 
into a 50 c.c. Erlenmeyer flask and the walls of the larger flask are rinsed 
with 20 c.c. of water, the water being measured in a cyhnder and approxi- 
mately a third used for each of three washings. When the solution and 
wash fluids, all measuring about 26 c.c, has been transferred to the flask, 
0.3 c.c. of a 0.1 per cent, solution of neutral red (dissolved in 50 per cent, 
alcohol) is added. 0.02 N carbonate-free NaOH is then run in from a buret 
until the color of the solution matches that of 29 c.c. of a standard piiosphate 
solution, of Pjj 7.4, contained in a similar 50 c.c. flask and to which 0.3 c.c. 
of the neutral red has been added. ^ In place of neutral red, 0.3 c.c. of a 0.04 
per cent, solution of phenolsulphonephthalein (phenol red) may be used as 
indicator, and gives an end-point slightly more easy to distinguish than that 
of neutral red. The standard, in the case of phenol red, must be a solution 
of Pjj 7.2, owing to the influence of the proteins on this indicator, as Homer 
has shown. 

It is desirable to use for the titration a micro-buret, although this is not 
absolutely essential though the results will be a trifle more accurate. A 
3 c.c. micro-buret is advised, divided into 0.02 c.c. divisions which are suffi- 
ciently separated to permit of readings of o.oi c.c. The tip is drawn out to a 
fine point, so that the drops are small, measuring about 0.03 c.c. each. 
However, in the absence of such a buret, an ordinary one with o.i c.c. gradu- 
ations will suffice. With both indicators, a peculiar phenomenon occurs as 
the end-point is approached. Each drop appears to change the color past 
the end-point, but within a few seconds the color shifts back, and it is seen 
that at least another drop is needed before the real end-point is reached. 
Consequently, the final color comparison should not be made until at least 
30 seconds after the last drop of 0.02 N NaOH has been added. 

Calculation. 

The number of c.c. of 0.02 N NaOH used in the titration is subtracted 
from the number required to neutralize to the same indicator 5 c.c. of the 
0.02 N HCl used. This number is, of course, approximately 5, but it usually 
varies slightly from that because of the calibration error of the 5 c.c. pipet 
used for measuring the acid. Consequently the maximum accuracy is 
obtained by performing a preliminary titration on 5 c.c. of the acid plus 20 

1 The standard solution of Ph 7- 2 and 7.4 may be made according to the directions given in 
the discussion of the colorimetric method for determining the hydrogen ion concentration 
of the blood, which follows this. It is important that the 0.02 N NaOH be protected from 
contact with atmospheric CO2 and from glass. It should, therefore, be kept in parafl&ned 
bottles and the buret filled with fresh solution as needed. In order to obtain a carbonate- 
free solution, one may dissolve the NaOH in an equal weight of water. The Na2C03 settles 
out of such a concentrated alkali solution and the supernatant liquid may be used as the 
basis of the dilution by taking 5.5 c.c. of this strong solution and diluting to 5 liters, stand- 
ardizing it against 0.02 N HCl using neutral red as an indicator. In performing this titra- 
tion it is advisable to run the acid into the alkali, titrating from the yellow alkaline color 
to the acid red. 



3.o6 c.c. 



428 DIAGNOSTIC METHODS 

C.C. of distilled water, using the same pipet, indicator, and end-point as in 
the plasma titration. For example, 

o . 02 N NaOH = HCl added 5 . 09 c.c. 

0.02 N NaOH taken in titration 2.03 c.c. 

0.02 M NaHCOa in 2 c.c. plasma or 1 

o.oi M NaHCOs in i c.c. plasma J 

3 . 06 -^ 100 = o. 0306 = molecular concentration of NaHCOs 

in plasma. 
3.06X22.4 (or 0.0306X2240) = 68.5 volume per cent. 
CO2 bound as bicarbonate in the plasma. 

Since the titration result represents c.c. of o.oi M NaHCOs per c.c. of 
plasma, it is transformed into terms of molecular concentration of NaHCOs 
in the plasma merely by dividing by 100. For the sake of comparison with 
results of bicarbonate determinations by the CO2 combining method, the 
molecular concentration is multiplied by 2,240, in order to obtain results in 
terms of CO2 per 100 c.c. of plasma. According to the gas laws, the amount 
of CO2 contained in a M carbonate solution is 22,400 c.c. per liter (measured 
as CO2 gas at 0° and 760 mm.) or 2,240 c.c. of gas per 100 c.c. of solution. 
Hence, multiplying the bicarbonate molecular concentration by 2,240 or 
multiplying the c.c. of 0.02 N acid used in the titration by 22.4, gives the 
volume per cent, of bicarbonate CO2 in the plasma. Inversely, dividing the 
volume per cent, of CO2 by 2,240 transforms the CO2 figures into terms of 
molecular concentration. 

The results with this method agree closely with those of the CO2 capacity 
method over the range of bicarbonate concentrations ordinarily encountered 
in man, even in severe acidosis. Below this range the titration continues 
to give accurate results, while the CO2 capacity method gives somewhat 
higher values. The normal value for the molecular concentration of bicar- 
bonate by this method is 0.0265, corresponding to a CO2 capacity of 61.6. 
Stillman recommends that the results be expressed in terms of millimolecular 
concentration (i millimolecular = 0.001 molecular).^ 

3. Methods of Determining Hydrogen-ion Concentration 

A. Electrometric Method. 

This method, known as the gas chain method, is the most accurate one 
at our command. However, it requires a delicate and expensive physico- 
chemical apparatus and, which is even more important, considerable special 
training in such methods before results may be satisfactory. The method 
consists in measuring the voltage (electric force )set up in a battery of which 
one electrode is pure hydrogen gas in intimate contact with the solution 
whose Ph it is desired to measure, and the other electrode is of known voltage, 
the more usual one being the calomel electrode. The development of the 

1 Van Slyke, Stillman, and Cullen, Jour. Biol. Chem., 1919, XXXVIII, 167; Stillman, 
Ibid., 1919, XXXIX, 261. 



THE BLOOD 



429 



electric force at the H electrode is dependent on the concentration of free H 
ions in the solution, which determines the rate of diffusion between the free 
H ions in the solution and the H electrode. As everything in the battery is 
constant, except the concentration of free H ions in the solution, the total 
electromotive force developed at the H electrode is proportional to the Ph 
of the unknown solution. While, as stated above, this method is one requir- 
ing much training in electrochemistry methods, yet it is to be said that the 
colorimetric methods, which will be discussed in detail, are somewhat less 
accurate, unless the solutions used are carefully checked by the electro- 
metric method. The literature contains quite a number of reports based 
on this method, yet it is not one that can find a place in the usual clinical 
laboratory, so that the details of the method will be omitted. 

B. Colorimetric Methods. 

This method of determining H ion concentration is based on the fact that 
each indicator has a characteristic zone of H ion concentration within which 
its color changes occur. In other words, the exact Ph at which indicators 
change in tint varies with the indicator employed. For convenience in using 
this method on other fluids than blood, the following table of Clark and Lubs 
is inserted. 

INDICATORS 



Chemical Name 



Common Name 



Color Change 



Range Ph 



Thymol-sulphon-phthalein 

Tetra - brom-phenol-sulphon-phtha- 

lein 

Ortho-carboxy-benzene-a z o-d i m e- 

methyl-aniline 

Ortho-carboxy-benzene-azo-d i p r o- 

pyl-aniline 

Dibrom-ortho-cresol-s u 1 p h o n-p h- 

thalein 

Dibrom - thymol-sulphon-phthalein. 

Phenol-sulphon-phthalein 

Ortho-cresol-sulphon-phthalein 

Thymol-sulphon-phthalein (alkaline 

range) ; 

Ortho-cresol-phthalein 



Thymol Blue 

Brom-phenol blue 

Methyl Red 

Propyl red 

Brom-cresol purple 

Brom- thymol blue 

Phenol red 

Cresol red 

Thymol blue 
Cresol phthalein 



Red-yellow 

Yellow-blue 

Red-yellow 

Red-yellow 

Yellow-purple 
Yellow-blue 
Yellow-red 
Yellow-red 

Yellow-blue 
Colorless-red 



1.2-2.8 

3.0-4.6 

4 . 4-6 . o 

4.8-6.4 

5.2-6.8 
6 . 0-7 . 6 
6.8-8.4 
7.2-8.8 

8.0-9.6 
8. 2-9-. 8 



Stock solutions of phenol red (or its monosodium salt) may be made of 0.6 
per cent, strength. The same strength of cresol red should be prepared. 
Methyl red and propyl red solutions are prepared by dissolving o.i gram in 
300 c.c. of alcohol and diluting to 500 c.c. with distilled water. The other 
indicators should be prepared, as stock, in a strength of 1.2 per cent. The 
strength, as used in the tests, is usually 0.02 per cent. 

Besides the indicators, it is necessary to have a set of "buffer" solutions 
whose Ph values have been accurately defined by electrometric methods. 
When these solutions, whose Ph is known, are treated with the indicator. 



430 DIAGNOSTIC METHODS 

certain colorations appear, which may be compared with those given by the 
solution to be tested, treated in the same way with the indicator. These 
buffer solutions must have such well defined compositions that they may be 
accurately reproduced with such Ph values as have been determined for 
the same solution by the gas chain method. Sorensen has suggested a set of 
these solutions, but these have the disadvantage that they are, in part, pre- 
pared with acetic acid and, as this is volatile, it becomes a difficult matter 
to reproduce an^xact solution. Other reagents suggested contain an uncer- 
tain amount of water o± crystallization, which does not permit of accurate 
determination of amount of salt to add to a solution in order to obtain a defi- 
nite concentration. For this reason, the writer adopts the set of salts 
advocated by Clark and Lubs, as it may be accurately standardized and aff- 
ords a wide range of Pjj values. 

The various mixtures are made up from the following stock solutions: 
M/5 potassium chlorid; M/5 acid potassium phosphate; M/5 acid potassium 
phthalate (KHC8H4O4); M/5 boric acid with M/5 potassium chlorid; 
M/5 sodium hydrate; and M/5 hydrochloric acid. The water used in the 
crystallization of the salts and in the preparation of the stock solutions and 
mixtures should be redistilled. So-called ''conductivity water," which is 
distilled first from acid chromate solution and again from barium hydroxid, 
is recommended. 

Ml ^ Potassium Chlorid Solution. 
The salt should be recrystallized three or four times and dried in an 
oven at about i2o°C. for two days. The solution contains 14.912 grams 
in I liter. 

M/ ^ Acid Potassium Phthalate Solution. 

Prepared by the method of Dodge (Jour. Ind. Eng. Chem., 1915, VII, 
29) modified as follows: Make up a concentrated KOH solution by dissolving 
about 60 grams of a high grade sample in about 400 c.c. of water. To this add 
50 grams of the commercial resublimed anhydrid of ortho-phthalic acid^ 
Test a cool portion of the solution with phenol phthalein. If the solution is 
still alkahne, add more phthalic anhydrid; if acid, add more KOH. When 
roughly adjusted to a slight pink with phenol phthalein add as much more 
phthalic anhydrid as the solution contains and heat till all is dissolved. 
Filter while hot, and allow the crystallization to take place slowly. The 
crystals should be drained with suction and recrystalHzed at least twice 
from distilled water. Dry the salt in platinum at 110-115 C. to constant 
weight. The standard solution should contain 40.82S grams of the salt to 
I liter of the solution. (The phthalic acid in the mother liquors may be 
recovered by acidifying these and purified by recrystallization.) 

M/ $ Acid Potassium Phosphate Solution. 
A high grade commercial sample of the salt is recrystallized at least 3 
times frorp distilled water and dried in platinum to constant weight at iio- 



THE BLOOD 43 1 

115 C. Solution should contain in i liter 27,232 grams. The solution 
should be distinctly red with methyl red and distinctly blue with brom 
phenol blue. 

ilf/5 Boric Acid with M/^ Potassium Chlorid. 
Boric acid should be recrystallized several times from distilled water. 
It should be air-dried in thin layers between filter paper and the constancy 
of weight established by drying small samples in thin layers in a desiccator 
over CaCl2. Purification of KCl is given above. One liter should contain 
12.4048 grams of boric acid and 14.912 grams of KCl. 

if/5 Sodium Hydroxid Solution. 
Dissolve 100 grams NaOH in loo c.c. distilled water in a Jena or Pyrex 
Erlenmeyer flask. Cover the mouth of the flask with tin foil and allow the 
solution to stand over night till the carbonate has mostly settled. Then 
prepare a filter as follows: Cut a hardened filter paper to fit a Buchner 
funnel. Treat it with warm strong (i : i) NaOH solution. After a few 
minutes decant the sodium hydroxid solution and wash the paper first with 
absolute alcohol, then with dilute alcohol and finally with large quantities 
of distilled water. Place the paper on the Buchner funnel and apply gentle 
suction until the greater part of the water has evaporated but do not dry so 
that the paper curls. Now pour the concentrated alkah upon the middle of 
the paper, spread it wdth a glass rod making sure that the paper, under gentle 
suction, adheres w^ell to the funnel, and draw the solution through with suc- 
tion. The clear filtrate is now diluted quickly, after rough calculations, to 
a solution somewhat more concentrated than N/i. Withdraw lo c.c. of 
this dilution and standardize roughly w^ith an acid solution of known strength, 
or with a sample of acid potassium phthalate. From this approximate stan- 
dardization calculate the dilution required to furnish an M/5 solution. 
Make the required dilution with the least possible exposure and pour the 
solution into a paraffined bottle to which a calibrated 50 c.c. buret and soda- 
lime guard tube has been attached. The solution should now be most care- 
fully standardized. Proceed as follows: Weigh out accurately on a chemical 
balance with standardized weights several portions of acid potassium phthal- 
ate of about 1.6 grams each. Dissolve in about 20 c.c. distilled w^ater and 
add 4 drops of phenol phthalein. Pass a stream of C02-free air through 
the solution and titrate wdth the alkah till a faint but distinct and permanent 
pink is developed. It is preferable to use a factor with the solution rather 
than attempt adjustment to an exact M/5 solution. 

Jf/5 Hydrochloric Acid Solution. 
Dilute a high grade of HCl to about 20 per cent, and distill. Dilute the 
distillate to approximately M/5 and standardize with the sodium hydroxid 
solution previously described. If convenient, standardize this, also, by the 
silver chlorid method. 



432 



DIAGNOSTIC METHODS 



Compositions of mixtures giving Ph values at 20 C, at intervals of 0.2 



Ph 
1 .0 
1 . 2 
1.4 
1.6 
1.8 
2.0 
2. 2 



4.0 
4.2 

4.4 
4.6 
4.8 
5-0 

5-2 

5-4 
5-6 
5-8 
6.0 
6.2 



5-8 
6.0 
6.2 
6.4 
6.6 
6.8 
7.0 
7.2 

7.4 
7.6 
7.8 
8.0 



o 
2 
4 
6 
8.8 
9.0 
9.2 
9-4 
9 
9 
10 



50 c.c. 
50 c.c. 
50 c.c. 
50 c.c. 
50 c.c. 
50 c.c. 
50 c.c. 
50 c.c. 
50 c.c. 
50 c.c. 
50 c.c. 
so c.c. 



50 c.c. M/5 KCl 
M/5 KCl 
M/5 KCl 
M/5 KCl 
M/5 KCl 
M/s KCl 
M/5 KCl 



50 c.c. 
50 c.c. 
50 c.c. 
50 c.c. 
50 c.c. 
50 c.c. 



Composition 
97.0 c.c. M/5 HCl 
64.5 c.c. M/5 HCl 

41.5 c.c. M/5 HCl 
26.3 c.c. M/5 HCl 

16.6 c.c. M/5 HCl 
10.6 c.c. M/5 HCl 

6.7 c.c. M/5 HCl 



Phthalate— HCl 
50 c.c. M/5 Phthalate 46 . 
50 c.c. M/5 Phthalate 39 . 
50 c.c. M/5 Phthalate 32 . 
50 c.c. M/5 Phthalate 26. 
50 c.c. M/5 Phthalate 20. 
50 c.c. M/5 Phthalate 14. 
50 c.c. M/5 Phthalate 9 . 
50 c.c. M/5 Phthalate 5 . 
50 c.c. M/5 Phthalate 2 . 



Mixtures 
70 c.c. M/5 HCl 
60 c.c. M/5 HCl 
95 c.c. M/5 HCl 
42 c.c. M/5 HCl 
32 c.c. M/5 HCl 
70 c.c. M/5 HCl 
90 c.c. M/5 HCl 
97 c.c. M/5 HCl 
63 c.c. M/5 HCl 



Dilute to 200 c.c. 
Dilute to 200 c.c. 
Dilute to 200 c.c. 
Dilute to 200 c.c. 
Dilute to 200 c.c. 
Dilute to 200 c.c. 
Dilute to 200 c.c. 

Dilute to 200 c.c. 
Dilute to 200 c.c. 
Dilute to 200 c.c. 
Dilute to 200 c.c. 
Dilute to 200 c.c. 
Dilute to 200 c.c. 
Dilute to 200 c.c. 
Dilute to 200 c.c. 
Dilute to 200 c.c. 



50 c.c. 
50 c.c. 
50 c.c. 
50 c.c. 
50 c.c. 
SO c.c. 
SO c.c. 
SO c.c. 
50 c.c. 
SO c.c. 
50 c.c. 
SO c.c. 



so c.c. 
50 c.c. 
50 c.c. 
so c.c. 
so c.c. 
so c.c. 
so c.c. 
so c.c. 
50 c.c. 
50 c.c. 
so c.c. 
so c.c. 



Phthalate— NaOH 
M/s Phthalate o . 40 
M/s Phthalate 3 . 70 
M/s Phthalate 7 . 50 
M/s Phthalate 12.15 
M/s Phthalate 17.70 
M/s Phthalate 23 . 85 
M/s Phthalate 29.95 
M/s Phthalate 35.45 
M/s Phthalate 39 . 85 
M/s Phthalate 43 . 00 
M/s Phthalate 45 . 45 
M/s Phthalate 47 . 00 



Mixtures 

M/sNaOH 
M/sNaOH 
M/sNaOH 
M/sNaOH 
M/sNaOH 
M/sNaOH 
M/sNaOH 
c.c. M/sNaOH 
c.c. M/sNaOH 
c.c. M/sNaOH 
c.c. M/sNaOH 
c.c. M/sNaOH 



Dilute 
Dilute 
Dilute 
Dilute 
Dilute 
Dilute 
Dilute 
Dilute 
Dilute 
Dilute 
Dilute 
Dilute 



to 200 c.c. 
to 200 c.c. 
to 200 c.c. 
to 200 c.c. 
to 200 c.c. 
to 200 c.c. 
to 200 c.c. 
to 200 c.c. 
to 200 c.c. 
to 200 c.c. 
to 200 c.c. 
to 200 c.c. 



KH2PO4— NaOH Mixtures 



KH2PO4 M/s 
KH2PO4 M/s 
KH2PO4 M/s 
KH2PO4 M/s 
KH2PO4 M/s 
KH2PO4 M/s 
KH2PO4 M/s 
KH2PO4 M/s 
KH2PO4 M/s 
KH2PO4 M/s 
KH2PO4 M/s 
KH2PO4 M/s 



3-72 

5-70 

8.60 

12 .60 

17.80 

23-65 
29.63 
35- 00 
39-50 

42.80 
45.20 
46.80 



c.c. M/sNaOH 
c.c. M/sNaOH 
c.c. M/sNaOH 
c.c. M/sNaOH 
c.c. M/sNaOH 
c.c. M/sNaOH 
c.c. M/sNaOH 
c.c. M/sNaOH 
c.c. M/sNaOH 
c.c. M/sNaOH 
c.c. M/sNaOH 
c.c. M/sNaOH 



Boric acid, 



M/s H3BO3, 
M/s HsBOj 



M/s 
M/s 



M/s H3BO3, M/s 



M/s H3BO3, 
M/s H3BO3, 
M/s H3BO3, 
M/s H3BO3, 



M/s 
M/5 
M/s 
M/5 



M/s H3BO3, M/5 



M/s H3BO3, 
M/s H3BO3, 
M/s H3BO3, M/s 
M/5 H3BO3, M/s 



M/s 
M/s 



-NaOH Mixtures 

M/sNaOH 
M/sNaOH 
M/sNaOH 
M/sNaOH 
M/sNaOH 
M/sNaOH 
M/sNaOH 
M/s NaOH 
M/sNaOH 
M/sNaOH 



KCl- 

KCl 2.61 c.c. 

KCl 3-97 c.c. 

KCl 5.90 c.c. 

KCl 8.50 c.c. 

KCl 12 .00 c.c. 

KCl 16.30 c.c. 

KCl 21 .30 c.c. 

KCl 26.70 c.c. 

KCl 32.00 c.c. 

KCl 36.85 c.c. 

KCl 40.80 c.c. M/sNaOH 

KCl 43.90 c.c. M/sNaOH 



Dilute to 
Dilute to 
Dilute to 
Dilute to 
Dilute to 
Dilute to 
Dilute to 
Dilute to 
Dilute to 
Dilute to 
Dilute to 
Dilute to 



Dilute to 
Dilute to 
Dilute to 
Dilute to 
Dilute to 
Dilute to 
Dilute to 
Dilute to 
Dilute to 
Dilute to 
Dilute to 
Dilute to 



200 c.c. 
200 c.c. 
200 c.c. 
200 c.c. 
200 c.c. 
200 c.c. 
200 c.c. 
200 c.c. 
200 c.c. 
200 c.c. 
200 c.c. 
200 c.c. 



200 c.c. 
200 c.c. 
200 c.c. 
200 c.c. 
200 c.c. 
200 c.c. 
200 c.c. 
200 c.c. 
200 c.c. 
200 c.c 
200 c.c. 
200 c.c. 



It is important to check the consistency of any particular set of these 
mixtures by comparing 5.8 and 6.2 phthalate with 5.8 and 6.2 phosphate, 
using brom cresol purple. Also, 7.8 and 8.0 phosphate should be compared 
with the corresponding borates using cresol red. 



THE BLOOD 



433 



In the method of Levy, Rowntree, and Marriott, which will be imme- 
diately discussed in detail, the set of standard solutions is made up from 
Sorensen's directions as follows: i/ 15 Macid or primary potassium phosphate, 
9.078 grams of the pure recrystallized salt (KH2PO4) are dissolved in freshly 
distilled water and made up to i liter; 1/15 M alkaline or secondary sodium 
phosphate. The pure recrystallized salt (Na2HP04.i2 H2O) is exposed to 
the air for from ten days to two weeks, protected from dust. Ten molecules 
of water of crystallization are given off and a salt of the formula Na2HP04. 
2H2O is obtained. 11.876 grams of this is dissolved in freshly distilled 
water and made up to i liter. The solution should give a deep rose red 
color with phenolphthalein. If only a faint pink color is obtained, the salt 
is not sufficiently pure. The above two 1/15 M solutions are mixed in the 
proportions indicated in the table below to obtain the desired Ph- 



Ph 


6.4 


6.6 


6.8 


7.0 


7-1 


7.2 


7-3 


7-4 


7-5 


7.6 


7.7 


7.8 


8.0 


8.2j 8.4 


KH2PO4 c.c. 


73 


63 


51 


37 


32 


27 


23 


19 


15.8 


13.2 


II 


8.8 


5-6 


3.2 2.0 




NaHP04.2H20 


27 


37 


49 


63 


68 


73 


77 


81 


84.2 


86.8 


89 


91.2 


94.4 


95.898.0 



A. Method of Levy, Rown-tree and Marriott. 

The principle of this method is based on the preceding considerations. 
The color of the standard solutions, to which the indicator has been added, 
is compared with that given by the solution to be tested after the addition 
of the indicator. While this method does not use the serum, as such, for 
the test, as it was believed that the coloring matters and proteins of the 
bloQd interfered with the proper colorimetric comparison, yet Homer has 
been able to obtain results, which agree closely with those of the electro- 
metric method, by using serum treated with neutral red as the indicator. In 
the method under discussion, the blood is dropped into collodion sacs and 
dialyzed for five minutes against 0.8 per cent, sodium chlorid solution. The 
dialysate, at the end of this period is free from proteins and coloring matter 
but these may make their appearance in the dialysate in from ten to twenty 
minutes. 

Preparation of Materials. 
I. Standard Color Tubes. 

The solutions are made as previously discussed, with the Ph values rang- 
ing from 6.4 to 8.4. Three c.c. of each of the solutions are placed in suitable 
small test-tubes (100X10 mm., inside measurement). Five drops of an 
aqueous o.oi per cent, solution of phenolsulphonephthalein (phenol red) 
are added to each tube. The tops are sealed off and the tubes are set aside 
for use as comparison colors. These colors may fade sHghtly in a month's 
time, but may still be used for comparison if less indicator is added to the 
"unknown" solution, as the color quality remains the same.^ 

^ If one does not wish to prepare these solutions for himself, he may obtain accurately 
standardized ones from Hynson, Wescott & Co., Baltimore, Md. 



434 DIAGNOSTIC METHODS 

2. Collodion Sacs. 

One ounce of celloidin (Anthony's negative cotton) is dissolved in 500 
c.c, of a mixture of equal quantities of ether and ethyl alcohol. The solid 
swells up and dissolves with occasional gentle shakings in forty-eight hours. 
As a small amount of brown sediment may separate out at first, the solution 
should stand for at least 3 or 4 days, after which time the clear supernatant 
solution is ready for use. If time permits, one may obtain better sacs from 
solutions that have "aged" for from 2 to 3 weeks. Small test-tubes (120 
by 9 mm. inside measurement) are filled with this solution, inverted, and half 
the contents poured out. The tubes are then righted, and the collodion 
allowed to fill the lower half again. A second time it is inverted and rotated 
on its vertical axis, the collodion being drained off. Care must be taken to 
rotate the tube, in order to secure a uniform thickness throughout. The 
tubes are clamped in the inverted position and allowed to stand for 10 min- 
utes, until the odor of ether disappears. They are then filled with cold 
water and allowed to soak for 5 minutes. A knife blade is run around the 
upper rim, so as to loosen the sac from the rim of the test tube, and a few 
c.c. of water are run down between the sac and the glass of the tube. By 
gentle pulling, the tube is extracted, after which it is preserved by complete 
immersion in water, to prevent becoming brittle and impermeable. 

3. Salt Solution. 

The salt solution, against which the blood is dialyzed, is an 0.8 per cent, 
solution, which must be free from acids other than carbonic. To determine 
this, a few^ c.c. of the salt solution are placed in a Jena test-tube and one or 
two drops of phenol red are added, whereupon a yellow color appears. On 
boiling, CO2 is expelled, and the solution loses its lemon color and takes on 
a slightly brownish tint. In the absence of this change, other acids are 
present, and the salt solution is not suitable for use. If, on the other hand, 
on adding the indicator, pink color at once appears, the solution is alkaline 
and cannot be used. 

Technic. 

One to 3 c.c. of clear (non-hemolytic) serum, plasma, whole or defibri- 
nated blood, are run, by means of a blunt pointed pipet, into a dialyzing sac 
which has been washed inside and outside with salt solution and which has 
been tested for leaks by filUng with salt solution. The sac is lowered into a 
small test-tube (100X10 mm., inside measurement) containing 3 c.c. of the 
salt solution, until the fluid on the outside of the sac is as high as on the in- 
side. From 5 to 10 minutes are allow^ed for dialysis. The collodion sac is 
removed and 5 drops of the indicator are thoroughly mixed with the dialy- 
sate. The tube is then compared with the series of standards until the cor- 
responding color is found, which indicates the Ph of the dialysate. For 
this color comparison a good light (natural or artificial) and a white back- 
ground are requisites. Readings must be made immediately. The tube 
matching most closely is selected and also the tubes on either side of it. 
These are critically inspected. A color falling between two of the standards 



THE BLOOD 435 

may be read, by interpolation, to another decimal place. For this com- 
parison, especially when the original material treated with the indicator is 
itself colored, the colorimeter of Walpole is essential. This consists of a 
block of wood with holes bored into the top for the test tubes and openings 
front and back through which the light may pass and, thus, permit com- 
parison of the color tints. The arrangement of the tubes in this colorimeter 
may be seen from the following diagram, lo c.c. of the fluid to be tested being 
usually employed instead of the 3 c.c. recommended by these workers. The 
extra tubes are added, in this comparison to equalize the color tints and 
make the results more accurate. 

Eye 
Left . Right 



10 c.c. solution 10 c.c. solution 

4 or 5 drops of indicator ■ No indicator 



ID c.c. water ■ 10 c.c. standard solution 

No indicator 4 or 5 drops of indicator 



Source of light . . 

By this method, oxalated blood from normal individuals gives a dialysate 
with a Ph varying from 7.4 to 7.6, while that of serum ranges from 7.6 to 
7.8. Variations from these figures toward the acid side (lowered Ph) are 
found in conditions evidenced by acidosis. Even in the more severe acidosis, 
however, the increase in H ion concentration becomes perceptible only in 
the final stages. It is, therefore evident that this value must be considered 
almost a constant and subject to variations under influence of the factors 
mentioned under the general discussion of the subject of acidosis.^ 

^ Levy, Ro\Yntree, and Marriott, Arch. Int. Med., 1915, XVI, 389; Levy and Rowntree, 
ibid., 1915, XVII, 525. In this connection see Sorensen, Biochem. Ztschr., 1909, XXI, 
.131 and 201; Walpole, Biochem. Jour., 1.910, V, 207; Hasselbalch, Biochem. Ztschr., 191 1, 
XXX, 317; Adler and Blake, Arch. Int. Med., 1911, VII, 479; Sorensen, Ergeb. Physiol., 
191 2. XII, 393; Hasselbalch and Lundsgaard, Biochem. Ztschr., 191 2, XXXVIII, 77; 
Lundsgaard, Ibid., XLI, 247; Hasselbalch, Ibid., 1913, XLIX, 451; Walpole, Biochem, 
Jour., 1913, VII, 260; Palmer and Henderson, Arch. Int. Med., 1913, XII, 153; Michaelis. 
Die Wasserstofi&onenkonzentration, Berlin, 1914; Walpole, Biochem. Jour., 1914, VIII, 
628; Jour. Chem. Soc, 1914, CV, 2501 and 2521; Peabody, Arch. Int. Med., 1914, XIV, 
236; Milroy, Quart. Jour. Exper. Physiol., 1914, VIII, 141; Hasselbalch and Gammel-, 
toft, Biochem. Ztschr., 1915, LXVIII, 206; Crozier, Rogers and Harrison, Surg. Gyn. and 
Obs., 1915, XXI, 722; Crile, Ann. Surg., 1915, XXXVII, 257; Lubs and Clark, Jour. 
Wash. Acad. Sc, 1915, V, 609; Clark and Lubs, Jour. Infect. Dis., 1915, XVII, i6c; Mar- 
riott, Arch. Int. Med., 1916, XVII, 840; Hurwitz, Meyer, and Ostenberg, Bull. Johns Hopk. 
Hosp., 1915, XXVII, 16; Lubs and Clark, Jour. Wash. Acad. Sc, 1916, VI, 481; de Corral, 
Biochem. Ztschr., 1916, LXXII, i; Scott, Jour. Lab. and Clin. Med., 1916, I, 608; Mc- 
Clendon, Jour. Biol. Chem., 1916, XXIV, 519; Clark and Lubs, Ibid., XXV, 479; McClen- 
don and Magoon, Ibid., 669; Parsons, Jour. Physiol., 1917, LI, 440; Clark and Lubs, Jour. 
Bacteriol., 1917, II, i, 109, and 191; Sonne and Jarlov, Hospitalstid., 1917, LX, 1247; 
CuUen, Jour. Biol. Chem., 1917, XXX, 369; Homer, Biochem. Jour., 1917, XI, 283; 
McClendon, Jour. Biol. Chem., 1918, XXXIII, 19; Barnett and Chapmann, Jour. A. M. A., 
1918, LXX, 1062; Macleod, Jour. Lab. and Clin. Med., 1919, IV, 315; Jones, Jour. Infect. 
Dis., 1919, XXV, 262; Fennel and Fisher, Ibid., 444; Kiigler, Jour. Bacteriol., 1919, IV, 35. 



436 DIAGNOSTIC METHODS 

B. Marriott's Method for AIkali Reserve. 

As has been stated, the alkaH reserve of the plasma is made up of bicar- 
bonates, alkah phosphates, and alkah protein compounds, all of these being 
present, under normal conditions, in fairly constant amounts. It is due to 
these constituents that the Ph of the blood is maintained at a constant value. 
As acid is introduced into the blood, a certain amount of this reserve is drawn 
upon and the reaction of the blood shifts towards the acid side. While 
the increased elimination of CO2 through the lungs may, for a time compen- 
sate for this, yet it does not remove the nonvolatile acids nor does this 
replenish the supply of alkali. Just so long as pulmonary ventilation com- 
pensates for the increased production of CO2 acidosis may not be evident, 
but sooner or later, the alkali reserve becomes so depleted that the condition 
may be measured by any of the methods previously discussed. 

The method of Marriott is a modification of the preceding one of Levy, 
Rowntree and Marriott. The changes introduced have to do, as far as the 
materials used in the test are concerned, with the preparation of the solu- 
tions used. The phosphate solutions, employed in the previous test, are 
made up in the same strength by Marriott, but, instead of diluting to i 
liter with water, the phosphates are dissolved in about 800 c.c. of water and, 
then diluted to i liter with 200 c.c. of o.oi per cent, solution of phenolsul- 
phenephthalein, thus obtaining a combination of the standard solution with 
the dye. These solutions are kept in Non-sol bottles and may contain a small 
crystal of thymol to prevent growth of molds. The sacks are prepared in the 
same way for both tests. The salt solution, in Marriott's method, is an 0.8 
per cent, solution in water, to which 220 c.c. of o.oi per cent, phenolsulpho- 
nephthalein solution are added and the whole made up to i liter. 
Technic. 

The determination should be made, in both these tests, in a room free 
from both acid and alkaline fumes. Exactly 0.5 c.c. of serum, oxalated plas- 
ma, or whole blood, is pipetted into one of the small collodion sacs, which 
has been previously washed inside and out with the salt solution (in this 
washing, no part of the sack except the top edge should be touched with the 
fingers; the sack may be emptied by tipping it with a clean glass rod or with 
a microscopic slide). This sack is l9wered into a small test-tube, approxi- 
mately 8 mm. internal diameter and 50 mm. long, centaining 2 c.c. of the 
indicator-salt solution. The level of the fluid on the outside of the sack 
should be at least as high as that on the inside. At the end of 7 minutes the 
sack is removed and the dialysate transferred to a clean test tube 100 to 140 
mm. long and having the same diameter as the tubes containing the phos-i 
phate standards. A rapid current of air is bubbled through the solution in 
order to remove the CO2. This is accomplished by means of an atomizer 
bulb connected with a narrow glass tube drawn out to a capillary point. 
The air current should be as rapid as possible without blowing liquid out of 
the test tube. Foaming rarely occurs, but if it should, add a drop of toluol. 
This blowing is continued for 3 minutes and then the color of the tube is 



THE BLOOD 437 

compared with the standard solutions in the manner previously discussed. 
For convenience of expression the reserve alkalinity is stated as RPh to 
differentiate it from the usual Ph- 

The serums of a large number of normal adults showed, in every instance, 
a RPh value of 8.5 + 0.05, provided the subjects were on a general mixed diet. 
After a fast of sixteen hours, the reading was 8.35. For normal infants, under 
I year of age, a value of RPh 8.3 was not infrequently noted. Values for 
the RPh oi from 8.4 to 8.55 correspond to an alveolar CO2 tension of from 
38 to 45 mm., and are to be considered as normal values for adults. Values 
between 8.0 and 8.3 correspond to an alveolar CO2 tension of from 28 to 35 
mm., and indicate a moderate degree of acidosis. When the value for 
RPh is as low as 7.7, corresponding to an alveolar CO2 tension of 20 mm., 
the individual is in imminent danger. Marriott states that it is his exper- 
ience that unless the RPh of the serum is below 7.9, the acidosis may be suc- 
cessfully combated by dietetic regulation or by administration of alkali by 
mouth. When it falls below 7.9, intravenous administration of alkali is 
usually indicated.^ 

As a measure of the depletion of the alkaline reserve of the system as 
a whole the method has been advanced of determining how much alkali may 
be administered in the form of NaHCOs to render the urine alkaline. Ob- 
viously, when the alkaline reserve is at or near the normal value, very little 
will be necessary. Sellards and Palmer and Henderson have shown that 
only 5 grams a day can be taken without making the urine alkaline, when 
conditions are normal. This quick response to intake of alkalis is seen in 
the so-called "alkaline tide" of the urine following meals. When the alka- 
line reserve is seriously depleted, large quantities, even as much as 125 
grams of bicarbonate daily may be taken without causing an alkaline reac- 
tion of the urine. Macleod states that "for practical purposes it is no doubt 
the best test of acidosis at present available in routine clinical work."^ 
(D) Specific Gravity. 

The specific gravity of normal blood varies between 1,055 and 1,065, 
the average being 1,060. Certain variations in this figure are observed de- 
pending on the sex or age of the patient or upon the time and temperature at 
which the determinations are made. 

^ Marriott, Arch. Int. Med., 1916, XVII, 840. In this connection, Henderson, Harvey 
Lectures, 1914-1915, X, 132; Henderson and Palmer, Jour. Biol. Chem., 1914, XVII, 
305; Ibid., 1915, XXI, 37; Begun, Herrmann, and Miinzer, Biochem. Ztschr., 1915, LXXI, 
255; Lang, Biochem. Jour., 1915, IX, 456; Macleod, Jour. Lab. and Clin. Med., 1916, 

II, 54; Marriott, Jour. A. M. A., 1916, LXVI, 1594; Howland and Marriott, Am. Jour. Dis. 
Child., 1916, XI, 309; Grulee, Ibid., 1917, XIII, 44; Underhill, Jour. A. M. A., 1917, LXV- 

III, 497; McEllroy, Jour. A. M. A., 1918, LXX, 846; Reimann and Bloom, Jour. Biol. 
Chem., 1918, XXXVI, 211; MacNider, Jour. Exper. Med., 1918, XXVIII, 501 and 517; 
Schwartz, Levin, and Mahnken, Jour. Cutan. Dis., 1919, XXXVII, 575; Asada, Am. Jour. 
Physiol., 1919, L, i; Blackader, Can. Med. Assoc. Jour., 1919, IX, 978; Van Slyke, Stillman, 
and Cullen, Jour. Biol. Chem., 1919, XXXVIII, 167; McClendon, von Meysenbug, Eng- 
strand, and King, Ibid., 539; Stillman, Ibid., XXXIX, 261; Buell, Ibid., XL, 29. 

2 Henderson and Palmer, Jour. Biol. Chem., 1912, XIII, 393; Ibid., XIV, 81; Ibid., 
XVII, 305; Sellards, Bull. Johns Hopk. Hosp., 1914, XXV, 141; Henderson and Palmer, 
Jour. Biol. Chem., 1915, XXI, 37; Frothingham, Arch. Int. Med., 1916, XVIII, 717; 
Sellards, Principles of Acidosis, Harvard Univ. Press, 191 7; Macleod, Jour. Lab. and 
Clin. Med., 1919, IV, 315. 



438 DIAGNOSTIC METHODS 

The most accurate method of determining the specific gravity is, of course, 
the use of the pycnometer. This method is open to the objection that it re- 
quires much more blood than can usually be obtained in routine work. In 
cases in which bleeding can be resorted to without detriment to the patient 
this method is the one to use, as it gives the most reliable and accurate results. 
The writer has used it in many cases of pneumonia, where the withdrawal 
of a certain amount of blood is often beneficial, and finds the results all that 
could be desired. . Besides the quantity of blood (5 to 50 c.c.) which is re- 
quired there is also necessary a very accurate chemical balance, else the 
results will be influenced by the inaccuracies in the weighings. 

The techuic is as follows: weigh the pycnometer empty, filled with dis- 
tilled water, and then filled with blood. Care should be taken to have the 
vessel absolutely dry and clean before weighing it empty and before filling 
with either water or blood. Subtract the weight of the empty bottle from 
that of the bottle filled with blood and divide this figure by the difference in 
weight between the bottle filled with water and the empty bottle. The result 
will be the specific gravity of the blood, water being taken as unity. We 
should be careful in this determination to have the temperature of the \/^ater 
the same as that of the blood in order to insure accurate results. 

Method of Schmaltz. i 

This method is a modification of the above and does not give quite as 
accurate results. It consists in the use of small tubes, which hold about 
i/io c.c. These tubes are constricted at the end to prevent loss of blood and 
are filled by capillary attraction. The determination is made in the same 
way as with the pycnometer. More or less manipulative skill is necessary 
in the handling of these tubes, but the results are sufficiently accurate for 
most purposes. 

The more frequently employed methods of determining the specific 
gravity are the so-called areometric ones. The principle of these procedures 
is the determination, by the use of accurate hydrometers, of the specific 
gravity of a liqi;id mixture, in the center of which a drop of blood will re- 
main suspended. 

Method of Hammer schlag. 

This method is a strictly areometric one and consists in the use of a 
mixture of benzol and chloroform into which a drop of blood is introduced 
through a capillary tube. If the drop rises in the mixture benzol is added 
and if it sinks chloroform is used. The point at which the drop of blood 
remains suspended in the center of the perpendicular axis of the mixture is 
taken as the one representing the specific gravity of the blood. As the means 
of estimating the density of this mixture, we employ an accurately gradu- 
ated hydrometer. It must be remembered that the fluid mixture should be 
well stirred after the addition of either benzol or of chloroform in order to 
insure uniform density throughout. As the mixture evaporates rapidly we 
must work quickly, and should confirm our results by a duplicate determina- 
tion. A further precaution should be to allow no bubbles of air to adhere 
to the drop. This is fairly well accomplished by the lise of the capillary 



THE BLOOD 



439 




blood pipet. It is also essential that the temperature of the mixture 
should not vary to any appreciable extent. This method is simple and, 
with the precautions mentioned, will yield good clinical results. 

The specific gravity of the serum may be tested in this same way, first 
allowing the blood to coagulate in sealed tubes and then drawing off a 
drop or two of the separa^ted serum. 

Naturally, the specific gravity of the blood is a measure of its concentra- 
tion .and, hence, of its water content. We are, therefore, certain to find 
variations in this factor under the influence of any physiologic or patho- 
logic changes, which are associated with fluctuations in the 
volume of blood. 

Physiologically, the specific gravity is higher in men 
-than in women and children; is higher in venous than in 
arterial blood, and is lower after ingestion of large quantities 
of fluid in the food or after infusions. Clinically, we find 
that the specific gravity of the blood runs parallel to the 
number of corpuscles and to the amount of hemoglobin in 
the ted cells. So striking is this ratio that it was formerly 
used to determine the percentage of hemoglobin in the 
blood. Any niarked alteration in these constituents gives 
rise to a variation in the specific gravity. Thus, there is 
observed in anemia in which there is a lowered percentage 
of hemoglobin, and in those forms of secondary anemia 
which are characterized more particularly by diminution in 
the number of cells, a low figure for th'"- specific gravity. 
In polycythemia, on the other hand, we find the specific gravity increased 
as a result of the increased corpuscular content of the blood. 

Pathologically, the specific gravity may run between 1,062 and 1,068. 
Ah increase is noted in practically all febrile diseases, in those conditions 
associated with cyanosis, and in disorders leading to obstructive jaundice. 
In conditions showing marked diuresis, diarrhea, or sweating an increase is 
likewise observed, but such changes are usually of slight duration, as the 
blood soon adapts itself to the condition by withdrawing liquid from the 
tissues to compensate for the loss in the above processes. In nephritis we 
may find either an increase or a decrease in the specific gravity, depending on 
the osmotic changes which take place in this disease. 
(£) Viscosity of the Blood. 

Freshly drawn blood has a greasy feeling, which is replaced by a stickiness 
as coagulation proceeds. This viscosity or internal resistance of the blood 
depends, to a large extent, upon the cellular content of the tissue and is 
distinct from the phenomenon of coagulation. 

Many methods have been advanced for the determination of this property 
of the blood, that of Determann^ being as clinically accurate and simple as 
any. So many uncertain factors influence the; viscosity that the writer feels 

^ Munch, med. Wchnschr., 1907, LIV, 1130; Die Viskositat des menschlichen Blutes., 
Wiesbaden, 19 10. 



Fig. 120. — -Pyc- 
nometer. 



440 DIAGNOSTIC METHODS 

that this determination can add little to our clinical knowledge. As Burton- 
Opitz^ says, "Although the results have been gratifying in so far as normal 
viscosity- values has been established for the human blood, it seems doubtful 
whether this method will ever be perfected in a way that small variations can 
be accurately and safely recorded. Nor do the limitations lie wholly in the 
method. One of the gravest obstacles, encountered in establishing slight 
differential values, exists in the variability of the viscosity itself." 

It has been shown that the degree of viscosity (n) is influenced by cold, 
withdrawal and application of heat, the former factors causing an increase, 
while the latter lowers it. Hirsch and Beck demonstrate that the lower the 
specific gravity of the blood the less marked is its viscosity. These results 
agree with those that indicate that the lower the specific gravity the lower the 
number of cellular elements. We may readily see, therefore, why the blood 
in anemia and leukemia shows such a slight tendency to become sticky or to 
form rouleaux. The researches of Rotky show that n varies between 5.02 
and 5.52 under normal conditions (water being i), is 1.69 in anemias, 3.34 
to 5.58 in nephritis, 13.56 in febrile states and 16.93 ^^ cyanotic conditions. 
According to Hess, the normal viscosity of male blood ranges between 4.3 
and 5.3, while that of female blood varies from 3.9 to 4.9. The normal 
relation^ 

Hemoglobin 

-^rr. r- = 17 tO 21. 

Viscosity ' 

(F) Coagulation of the Blood. 

It is impossible in this place to discuss the physics and chemistry of the 
process of coagulation, more than to say that this phenomenon is due to the 
conversion of the fibrinogen of the plasma into fibrin. This change takes 
place under the influence of a ferment, thrombase, which is present in the 
leucocytes and platelets in the form of prothrombase. This latter zymogen, 
through the influence of calcium compounds, is changed into thrombase, the 
active agent in bringing about coagulation. 

It is occasionally of clinical importance to know the time of coagulation 
of the blood under certain conditions, as one of the normal processes of this 
tissue, when outside the vessels, is coagulation. This process takes place 
normally in from two to eight minutes, depending on several factors, among 
which are the length of time the blood is in contact with the tissues, the depth 
of the incision, the pressure with which the blood is expelled, the nature of the 
vessel into which the blood flows, and the temperature at which coagulation 
takes place. 

It has been found that certain variations in the normal coagulabiUty are 
present at different hours of the day and are observed when blood is drawn 
from different parts of the body.^ 

^ Jour. Am. Med. Assn., 1911, LVII, 353. 

2 Austrian, Bull. Johns Hopkins Hosp., 191 1, XXII, 9. See, also, Matsuo, Deutsch. 
Arch. f. klin. Med., 191 2, CVI, 433; Langstroth, Jour. Exper. Med., 1919, XXX, 597 and 
607. 

2 See Cohen, Arch. Int. Med., 191 1, VIII, 684 and 820; Howell, Am. Jour. Physiol., 191 1, 
XXIX, 187; Therap. Gaz., 191 2, XXXVI, 95; Bordet and Delange, Ann. de I'lnst. Pasteur, 



THE BLOOD 441 

Dorrance^s Method. 

This method^ is simple and reliable. The instrument consists of an 8- 
ounce thermos bottle without the silver lining, an aluminium stand and two 
rubber stoppers, one of which has one hole for the passage of a thermometer, 
while the other bears four holes each of which is lined with a brass flange. 
Into each of these holes fits a glass rod 4^^-^ inches long and 8 mm. in diame- 
ter. The lower end of each rod is cone-shaped, the fiat tip being 4 mm. in 
diameter. The other end is slightly bulbous to hold the rod in place. 

Fill the bottle with water at 98°F. to within an inch of the top. Insert the 
stopper with thermometer. Scrub the glass rods with soap and water and 
cleanse them with alcohol and ether. Substitute the stopper with rods for 
the other^ so as to warm them to gS'^F. Puncture the ear or finger and wipe 
away the first two or three drops of blood. Remove the stopper with rods, 
wipe the latter dry and touch the drop of blood with the tip of each rod in 
turn, getting as nearly as possible the same amount of blood on each. Note 
the time and replace the stopper in bottle. 

The end-point is determined in three ways: (i) At the end of two and one- 
half minutes push rod i down into the water. All the blood normally falls off 
the end and breaks up into a fine cloud. At intervals of one minute the other 
rods in turn are pushed into the water. Some blood falls off but the particles 
become coarser, the amount remaining on the tip becoming greater until 
with rod 4 most of the blood remains, if coagulation has occurred. If no 
coagulation obtains, the test is repeated with larger intervals between the 
immersions in water. (2) Remove stopper with rods and hold up to the 
light. The tips will show more or less of a red color depending on the degree 
of coagulation, rod i being clear while rod 4 is almost covered with a red clot. 
(3) Touch the tip of each rod to filter paper. The depth of color of the blot 
will indicate the degree of clotting. The average coagulation-time by this 
method is three and one-half to five and one-half minutes. 

Rudolf's Method. 

This^ is a modification of the older one of Sabrazes^ and applies some of the 

1912, XXVI, 657; Berl. klin. Wchnschr., 1914, LI, 497; Barratt, Jour. Path, and Bacteriol., 

1913, XVII, 303; Whipple, Arch. Int. Med., 1913, XII, 637; Cannon and Gray, Am. Jour. 
Physiol., 1914, XXXIV, 232; Cannon and Mendenhall, Ibid., 225, 243 and 251; Gray and 
Lunt, Ibid., 332; Howell, Ibid., 1914, XXXV, 143 and 474; Pepper and Krumbhaar, Jour. 
Infect. Dis., 1914, XIV, 476; Fingerhut and Wintz, Munch, med. Wchnschr., 1914, LXI, 
363; Stuber and Heim, Ibid,, 1661; Fonio, Mitt. a. d. Grenzgeb. d. Med. u. Chir., 1914, 
XXVII, 642; Ibid., 1914, XXVIII, 313; Lee and Vincent, Arch. Int. Med., 1914, XIII, 
398; Ibid., 1915, XVI, 59; Hurwitz and Drinker, Jour. Exper. Med., 1915, XXI, 401; 
Drinker, K. R. and C. K., Am. Jour. Physiol., 1915, XXXVI, 305; Mendenhall, Ibid., 1915,^ 
XXXVIII, 33; Denny and Minot, Ibid., 233; Bloch, Arch. d. Mai. du Coeur, i9i5,VIII, 249 ;' 
Barratt, Biochem. Jour., 1915, IX, 511; Minot, Jour. Med. Res., 1915, XXXIII, 503; 
Herzfeld and KHnger, Biochem. Ztschr., 1915, LXXI, 391; Klinger, Cor. Bl. f. Schweiz. 
Aerzte, 1915, XLV, 1622; Dale and Walpole, Biochem. Jour., 1916, X, 331; Hekma, 
Biochem. Ztschr., 1916, LXXIII, 370 and 428; Ibid., LXXVII, 249; Minot, Denny and 
Davis, Arch. Int. Med., 1916, XVII, loi; Stoldt and Vaughan, Jour. Lab. and Clin. Med., 
1916, I, 257; Lenoble, Bull, de 1' Acad, de Med. Paris, 1916, LXXVI, 365; Arch, des Mai. 
du Coeur, 1916, IX, 539. 

1 Am. Jour. Med. Sc, 1913, CXLVI, 562. See also, Lyon, Jour. A. M. A., 1916, LXVI, 
891. 

2 Am. Jour. Med. Sc, 1910, CXL, 807; 191 1, CXLII, 481. 
^ Folia Haemat., 1904, I, 394. 



442 DIAGNOSTIC METHODS 

points of the method of Kottmann.^ It is so simple and clinically reliable 
that the writer advocates it for general use. Thin glass tubes 1.5 mm. in 
diameter and about 18 cm. (7 inches) in length are used. A pint Thermos 
bottle is employed, its ordinary cork being replaced by a triply perforated 
rubber stopper. In these perforations rest two brass tubes 7 inches long and 
just large enough in caliber to hold easily the glass blood- tubes. The third 
perforation contains a thermometer. The apparatus rests on its side in such 
a way as to prevent its rolling about. The bottle is filled with water at 
2o°C. and the stopper is inserted. 

The finger or lobe of the ear is punctured, the exact time of puncture being 
noted. Two glass tubes are partially filled from the same drop, the blood 
being made to run nearly to the far end of each tube, which is placed, as filled, 
in a brass tube of the thermostat. The protruding ends, the ones at which 
the blood entered the tubes, are then sealed with a spirit lamp. In about five 
minutes the first tube filled is drawn out of its holder by the left hand (covered 
with a glove to diminish the effect of heat of the fingers), is touched with a 
sharp file, broken across and the broken ends slowly separated. The tube is 
at once replaced in the thermostat. This technic is carried out with the first 
tube at intervals of 15 to 30 seconds, until a thread of fibrin appears between 
the broken ends. The time between the puncture and the appearance of the 
fibrin is the "coagulation time" of the blood. Tube No. 2 may then be used 
as a control. Normally the coagulation time averages eight and one-half 
minutes by this method. 

Method of Boggs. 

This is a modification of the older method of Russell and Brodie and gives 
a lower coagulation time than does the preceding method. It is based on the 
fact that the corpuscles, set in motion by a current of air in a moist chamber, 
move freely and independently of one another at first but, as coagulation pro- 
ceeds, clumping of the cells occurs, and finally an elastic radial motion of these 
cells obtains. This is observed under the low power of the microscope. The 
writer refers elsewhere for the technic (see cut). 

Normally, the coagulation time, as evidenced by the formation of fibrin, 
is between two and nine minutes. ^ Anything above nine minutes means 
delayed coagulation. The importance of testing this function of the blood is 
observed more particularly in cases of suspected hemorrhagic diathesis, in 
which the period of coagulation is remarkably increased. In some cases of 
hemophilia it requires fifty minutes, while in certain of the purpuras from 10 
to 15 or more (Emerson). Likewise, it is customary to test this factor in 
cases of long-standing jaundice, in which surgical intervention for obstructive 
lesions of the biliary passages is to be undertaken, as here also the time is 
much increased. Moreover, we find the coagulation time delayed or imper- 
fect in cases of hemoglobinemia, asphyxia, and general dropsy.^ Poisoning 

^ Ztschr. f. klin. Med., 1910, LXIX, 415. 

2 Carpenter and Gittings (Am. Jour. Dis. Child., 1913, V, i) show that the average time 
for children is slightly longer than for adults. 

^ See Kreiss, Inaug. Dissert., Heidelberg, 1912; Nel, Inaug. Dissert., Berlin, 1912; Lands- 
berg, Biochem. Ztschr,, 1913, L, 245; Lee and White, Am. Jour. Med. Sc, 1913, CXLV, 



THE BLOOD 



443 



from the bite of certain serpents, such as the cobra, is characterized by a 
greatly delayed coagulation of the blood as well as by hemolysis. On the 
other hand, we notice a quicker coagulation in conditions associated with 
stasis, repeated hemorrhages, transfusion, hunger, and also under the 
therapeutic use of calcium chlorid and of gelatin. 

The formation of fibrin, as the end-product of coagulation, is increased 
in some cases and diminished in others.^ An increase in the amount of fibrin 
in the blood (hyperinosis) is observed in acute inflammatory processes and in 





Xathar.aC Vljl» 



Fig. 121. — Boggs' coagulometer: a, moist chamber; b, tip of tube through which air 
passes; c, cover which fits over moist chamber and which holds glass cone; d, pin-holejfor 
escape of air; e f, cross section of cover c; g, tip of glass cone upon which is placed the 
drop of blood. 



most infectious diseases. Hay em states that the density of the fibrin network, 
observed when blood is allowed to dry in thick smears on a glass slide, indi- 
cates the degree of resisting power of the individual against disease. The 
largest amounts of fibrin are observed in such conditions as pneumonia^ and 
acute articular rheumatism, but are seen to a lesser extent in parenchymatous 
inflammation, in inflammations of the mucous membranes and the skin, in 
the febrile stages of chronic suppurations, in hepatitis, influenza, diphtheria, 
acute gout, and erysipelas. A decrease in the amount of fibrin (hypinosis) 
may be observed in malignant growths, malarial fever, pernicious anemia, 
leukemia, and purpura. In parenchymatous nephritis the amount of fibrin 
is but slightly if at all increased, while in interstitial nephritis the increase 
may be notable (Da Costa). 

(G) Osmotic Pressure and Cryoscopy. 

The cryoscopic examination of the blood may be of some importance in 
the diagnosis of certain conditions, particularly in reference to the sufficiency 

495; Jaffe, Folia Haemat., 1913, XV, 167; Steiger, Wien. klin. Wchnschr., 1913, XXVI, 1749; 
Kahn, Am. Jour. Dis. Child., 1916, XI, 103; Hess, Arch. Int. Med., 1916, XVII, 203; 
Hurwitz and Lucas, Ibid., 543; Corachan and Mones, Siglo. Med., 1919, LXVI, 935 and 960. 

^ See Bosworth, Jour. Biol. Chem., 1915, XX, 91. 

2 Dochez (Jour. Exper. Med., 191 2, XVI, 693) finds the coagulation time lengthened in 
acute stage of pneumonia. Anders and Meeker, Jour. A. M. A., 1916, LXVII, 1591; 
Burns and Young, Am. Jour. Med. Sc, 1917, CLIV, 797. 



444 



DIAGNOSTIC METHODS 



I 



or insufficiency of the kidneys as regards the elimination of the urinary sohds. 
While this subject has not yielded as much information as was expected of it, 
a brief discussion seems essential. For a theoretical discussion of osmotic 
pressure and cryoscopy, I must refer the reader to 
works on physical chemistry. 

By cryoscopy from the Greek kryos, frost, and 
skopeo, to see, is meant the determination of the 
freezing-point of a solution and the referring of this 
figure to the freezing-point of the solvent, which is 
regarded as o. Substances in solution lower the 
freezing-point of the solvent in direct proportion 
to their molecular (dissociated or nondissociated) 
concentration. In determining the osmotic pres- 
sure of a solution, and cryoscopy is one of such 
methods, it is important to remember that the pro- 
teins have practically no osmotic value. We have, 
therefore, in this cryoscopic method a means of 
ascertaining directly the molecular concentration of 
the body fluids. 
^ rd I II y^ >k ^^^ determination of the freezing-point of the 

I 'fe-^-^^^— -^^ blood is best made by means of the Beckmann ap- 
pg^j.^^^g^ which may be found in works on physical 
chemistry. As the osmotic pressure of the serum is 
equal to that of the plasma or of the whole blood, 
the serum is generally used for this determination. 
Withdraw from a vein, preferably the median bas- 
ihc, about 30 c.c. of blood and allow it to coagulate 
in a clean closed vessel. Place the serum in the freez- 
ing tube of the Beckmann apparatus, adjust the 
thermometer and stirring rods, and proceed as 
directed elsewhere. 

The freezing-point of the blood is usually desig- 
nated as the small Greek delta (5), while that of the 
urine is given the sign A. Normally, blood freezes 
at — o.56°C., distilled water freezing at o. This point is subject to more or 
less physiologic variation depending upon the changes which occur in the 
blood after meals, exercise, baths, etc. This physiologic variation is transient 
and slight because the kidneys soon regulate the osmotic pressure (molecular 
concentration) of the blood by withdrawal of the constituents which have 
caused the abnormal tension. This close relationship between the kidneys 
and the blood is of great importance in the study of renal insufficiency, as 
many products of metabolic activity remain in the blood in cases of renal 
disturbance. This is more particularly true of the inorganic constitutents of 
the blood, as the organic elements do not materially affect its osmotic pro- 
portions. It is in those cases of renal insufficiency with a tendency to uremia 
that the most is to be expected from the cryoscopic examination of the blood, 




Fig. 122. — Beckmann 
apparatus. {Long.) 



THE BLOOD 445 

yet we find that Schoenborn/ in the study of 88 cases, observed practically 
normal figures for the cryoscopic point of the blood. Engelmann,^ on the 
other hand, reported a series of 36 cases in which the freezing-point averaged 
— o.664°C. It is true that in certain surgical conditions of the kidneys a 
parallelism is noticed between the lowering of the freezing-point and the 
development of the uremic symptoms. In cyanotic conditions of any origin 
whatever we find the cryoscopic point lower than the normal, owing to the 
fact that the CO2 is increased in amount in such cases. If the CO2 be driven 
off, the freezing-point becomes normal. Hence we see why, in certain cases of 
uncompensated cardiac disease, the freezing-point has frequently been re- 
ported much lower than the normal standard. There is practically no. 
disease of the blood itself about which a cryoscopic study will furnish any- 
information. 

From these considerations, as well as from many others, which I have not 
enumerated, the study of the cryoscopy of the blood does not seem to the 
writer of sufficient importance to warrant its adoption as a routine method 
of blood examination. It is, certainly, of no value whatever to the general 
practitioner who does not have access to a thoroughly equipped laboratory. 
The time consumed in such examinations would be much better spent in a 
more thorough study of the fresh and of the stained specimens. While 
cryoscopy may give some idea of the osmotic activity of the kidneys, it adds 
to blood examinations a slightly enlarged laboratory record, whose interpre- 
tation is a matter of more or less difficulty and whose results rarely ever give 
any information, beyond that which may be more easily and more readily 
learned by other methods. It is a purely theoretical method of following 
the metabolic activity of the system and reveals no signs of an oncoming 
uremia nor does it show us anything regarding the results of therapeutic 
measures. 

Naturally, variations in the osmotic pressure of the blood are of import- 
ance in the study of conditions associated with effusions into the various 
serous cavities, as such exudations are markedly influenced by the molecular 
concentration of the blood. For a discussion of such phases of the subject I 
must refer the reader to works on pathology. 

(H) Electric Conductivity. 

In the effort to enlarge the scope of blood examinations, the clinician and, 
more particularly, the laboratory worker have taken advantage of every- 
thing offered for the furtherance of their aim. In so doing they have of ter^ 
over-stepped themselves and complicated the examinations by useless ad-- 
ditions to their, technic. 

By electric conductivity is meant the reciprocal of the resistance, which 
a certain amount of solution offers to the passage of an electric current of 
known strength between two platinum electrodes of given size and a given, 
distance apart. This method is merely a measure of the number of elec- 

^ Wiesbaden 1904. 

- Mitt., a. d. Grenzg. d. Med. u. Chir., 1903, XII, 396. See, also, Krotoszyner and 
Hartman, Jour. A. M. A., 1913, LX, 188; Ibid., 1915, LXV, 1788. 



446 DIAGNOSTIC METHODS 

trolytes (both dissociated and nondissociated) in solution and is not affected 
by the nonelectrolytic organic substances of the blood, although, according to 
Hardy, ^ the proteins move to one or the other pole of the battery depending 
on the reaction of the fluid. The method usually adopted for estimating 
this factor is that of Kohlrausch, the resistance being balanced on a Wheat- 
stone bridge against a rheostat, the point of equilibrium being determined by 
means of a telephone attachment. 

As this determination can give us no information regarding the retention 
of organic products in the system, it is of comparatively little value in the 
study of metabolic or of blood diseases. It is in those conditions which are 
associated with retention of inorganic constituents that this method should 
be of value, and yet we find, according to the work of various authors, that in 
advanced nephritis, in which the retention of chlorids has been assumed, the 
conductivity of the serum is not increased to any extent, although the freezing- 
point is lower than the normal standards. This proves, its seems to the 
writer, that in such cases the substances capable of increasing the conduc- 
tivity — that is, the chlorids — are either not retained in the blood or are in 
such a combination as not to influence the conductivity of this tissue. 
We must, therefore, assume that the lowering of the freezing-point ob- 
served in such conditions is due more to the organic than to the inorganic 
constituents of the blood. As this determination is, in the writer's opin- 
ion, of so little practical value in blood work, a detailed description of the 
methods and results of observations will not be taken up. 

(6) Chemical Properties. 

The chemical examination of the blood resolves itself into a consideration 
of the composition of the whole blood, of the plasma, of the serum, and of 
the various cellular elements. Is is evident that the composition of the whole 
blood will depend upon the relations in which the single constituents of the 
blood stand to one another. ^ Naturally, physiologic influences are of great 
importance in the consideration of the composition of the fluid portion, while 
the cellular elements are less affected by these changes. In view of the 
recent work on opsonins and immunity, we must conclude that the chem- 
ical examination of the fluid elements of the blood may yield much impor- 
tant information as soon as proper methods of research are evolved. . 

Normally, the composition of the cellular elements remains almost con- 
stant and is but slightly affected by the varying composition of the fluid por- 
tions. As is well known, the corpuscles act more or less like semipermeable 
membranes, although Kahlenberg would have us believe that no such a con- 
dition is possible. At any rate, the membrane surrounding the erythrocyte 
allows the passage of certain inorganic and organic constituents of the plasma 
into the cells and permits the back-passage of certain constituents of the cells 
into the plasma. This question is intimately related to the subject of hem- 
olysis, which will be discussed in detail later. It is true that a many-sided 

^ Proc. Royal Soc, 1900, LXVI, 95. 

2 See Bonninger, Ztschr. f. exp. Path. u. Therap., 191 2, XI, i; also, Veil, Deutsch. Arch, 
f. klin. Med., 1914, CXIII, 226; Lowy, Deutsch. Arch. f. klin. Med., 1915, CXVII, 79. 



THE BLOOD 



447 



^x change exists between the red cells and the hypertonic plasma in which they 
float, such an exchange accounting very often for the alterations in the volume 
Of the corpuscles and frequently being the cause of abnormally developed 
Cells. This exchange is subject to certain restrictions. Thus we find that the 
Corpuscles show a very high content in potassium salts and a very low sodium 
Content, while the reverse conditions obtain in the serum. If free exchange 
^ook place between the cells and serum no such conditions could exist. It is 
known that potassium salts are closely associated with phenomena of growth 
and hence it might be possible to prove that cells, which are morphologically 
the older, show a less potassium content than do the newly developing ones. 
It is evident that a quantitative analysis of the blood can be of only com- 
parative value as far as the blood as a whole is concerned. We should ascer- 
tain on one side the relationship of the plasma and blood-corpuscles to each 
other, and on the other side the composition of each of these two chief con- 
stituents. As there are many difficulties in the way of such determinations, 
we will not go into detail regarding the chemical composition of the different 
portions of the blood, but will discuss the blood as a whole. According to C. 
Schmidt, the composition of the blood is as outlined in the following table. 



Man's. 

Corpuscles 513 02 

Water ^. 

Solids 

Hemoglobin, proteins, and 
other organic bodies . . . 

Inorganic bodies 

K2O 

NasO 

CaO ] 

MgO . . [ 

FesOs J 

CI 

P2O5 

Man's. 

Serum . 486.98 

Water 

Solids 

Protein and other organic bodies 

Inorganic bodies 

K2O 

NaaO 

CaO \ 

MgO J 

CI 

P2O5 



Woman's. 






396.24 




349 • 690 




272.560 


163.330 




123 .680 


159-590 




120.130 


3 -740 




3-550 


1.586 




1 .412 


0.241 




0.648 



0.320 



0.485 



0.898 




0.362 


0.695 




0.643 


Woman's. 






603 . 76 




439.020 




551-990 


47.960 




51.770 


43.820 




46 . 700 


4.140 




5.070 


0.153 




0.200 


1. 661 


' 


1. 916 


0.533 




0.608 


1.722 




0.144 


0.071 




2.202 



448 DIAGNOSTIC METHODS 

This table, while giving the composition of the whole blood, may readily serve 
as one from which the composition of the corpuscles and serum may be ob- 
tained by reducing the constituents to parts per i,ooo of either corpuscles or 
serum. It is beyond the scope of this work to discuss the chemical properties 
of the blood or of its constituents in detail, but certain points which have 
clinical interest must be taken up. 

The large excess of chlorin in the serum of man as compared with that 
of woman and the excess of phosphoric acid in the serum of woman as com- 
pared with that of man is noteworthy. These variations may later be shown 
to have a great influence upon sexual differences as regards metabolism.^ 

{A). Total Solids. 

The determination of the total solids and, hence, of the water content 
of the blood is often of importance, especially in cases of anemia.^ The method 
is as follows: Allow about i c.c. of blood to flow onto one of two previously 
weighed matched watch-glasses. Cover this with its mate and weigh them 
while in the moist condition. Separate the glasses and place them in a des- 
iccator over CaCl2 or H2SO4 for twenty-four hours, at the end of which time 
weigh them as before. The blood should have dried to a hard glassy mass 
in this time. The loss of weight will represent the water of the blood taken, 
from which amount the percentage may be calculated. Gumprecht and 
Stintzing and Biernacki advise the use of higher than room temperature, the 
former using 67°C. in an incubator, while the latter employs a heat of 100 
to i2o°C. Drying by these latter methods is often associated with loss; 
hence the writer is accustomed to use room temperature (about 2o°C.). 

Working in this way, many have found the dry residue (total solids) 
of the blood to be from 21 to 22.5 per cent., the water content being 77.5 to 
79 per cent. This figure shows marked variation under the influence of such 
processes as diarrhea, excessive sweating, and exudation into serous cavities. 
In anemia the solids are much reduced, while in leukemia they are increased. 

(B) Blood Pigments. 
Hemoglobin. 

The normal color of the blood is due to the presence of the pigments, 
hemoglobin and oxyhemoglobin. Usually the former is not large in amount, 
while the latter is the predominant factor in the color. In blood after as- 
phyxia we find a mixture of hemoglobin, pseudohemoglobin, and parhemo- 
globin; in arterial blood large amounts of oxyhemoglobin, and in venous 
blood a mixture of hemoglobin and oxyhemoglobin. 

Hemoglobin belongs to the class of bodies known as " chromoproteins " 
and, owing to its power of combining with various gases and thus aiding in 
the gaseous exchange of the body, may be styled a "respiratory protein." 
It is easily decomposed into a protein, globin (about 96 per cent.), and a pig- 
ment, hemochromogen (4 per cent.), which contains iron and is easily changed 

^ See Gettler and Baker (Jour. Biol. Chem., 1916, XXV, 211) for extensive analysis 
of the blood of 30 normal cases. Also Gettler and George, Jour. A. M. A., 1918, LXXI, 
2033. 

2 See Busch, Ztschr. f. exper. Path. u. Therap., 1913, XIV, 335. 



THE BLOOD 



449 



in the presence of oxygen into hematin. The iron content of hemoglobin 
is the portion which enables this pigment to exert its peculiar vital power of 
oxygen transference.^ 

The amount of hemoglobin in normal blood is variable, depending on 
the age of the subject examined.^ Normally, loo c.c. of adult human blood 
contain from 13 to 14 grams of hemoglobin (the average being about 13.77), 
which amount is not a constant factor at all ages. From the following table 
of Leichtenstern it will be seen that a definite age curve exists for this 
substance. 



Age. 


Grams per 100 c.c. of blood. 


I to 4 days, 


19.329 to 21.160 


8 to 14 days. 


17.869 to 16.124 


8 to 20 weeks. 


15.362 to 12.928 


6 months to 5 years, 10.971 to 1 1-373 


5 to 15 years. 


II. 151 to 11.796 


15 to 25 years. 


13.034 to 13.870 


25 to 45 years, 


14.727 to 15.013 


45 to 60 years, 


12.484 to 13.150 


Over 60 years. 


14.790 



Under certain conditions the hemoglobin is dissolved out from the red 
cells, leaving only their stromata behind. This gives rise to the condition 
known as hemoglobinemia which will be discussed in a later section. Accord- 
ing as hemoglobin is free or combined with certain gases, we have in the blood 
various derivatives of this pigment, each one of which has certain character- 




FiG. 123. — Direct- vision spectroscope. 

istics and a definite clinical importance. These derivatives are recognized by 
quahtative tests, especially by spectroscopic methods, and will be presented 
here; while the important tests used for the recognition of hemoglobin, either 
in the fresh state or as dried stains, will be taken up under the medicolegal 
discussion.^ 

Hemoglobin also called reduced hemoglobin, is much more soluble than 
oxyhemoglobin, its solution in water being more violet or purpHsh than one 
of oxyhemoglobin of the same concentration. Such solutions of hemoglobin 
absorb the blue and violet rays of the spectrum to a less marked degree than 

1 See Barcroft (Respiratory Function of the Blood, Cambridge, 1914) and Macleod, 
Physiology and Biochemistry in Modern Medicine, St. Louis, 1918. 

2 See Williamson (Arch. Int. Med., 191 6, XVIII, 505) for a spectrophotometric analysis 
of 919 cases of varying ages. His figures show that between the ages of 16 and 60 
there is a marked difference between the two sexes, this difference growing less after the 
60th year. The variations in the normal values for hemoglobin are greatest from birth to 
the i6th year. See; also, Appleton, Jour. Biol. Chem., 1918, XXXIV, 369. 

3 See Maestre and Lecha-Marzo, Arch, internat. de med. leg., 1914, V, 49. 



450 DIAGNOSTIC METHODS 

do those of oxyhemoglobin, but they strongly absorb the rays lying between 
C and D. In proper dilution a solution of hemoglobin shows a spectrum 
with one broad not clearly defined band between D and E, lying toward the 
red end of the spectrum a little over the Fraunhof er line D (see plate) . 

Pseudohemoglobin. 

According to Ludwig and Siegfried, blood, reduced by hyposulphites 
or by a stream of hydrogen to such an extent that the spectrum of oxyhemo- 
globin disappears, yields large amounts of oxygen when exposed to a vacuum. 
This loose combination of hemoglobin and oxygen, which gives the spectrum 
of hemoglobin, is called psuedohemoglobin. Hammarsten considers it an 
intermediate body between hemoglobin and oxyhemoglobin. 

Oxyhemoglobin. 

Oxyhemoglobin, also called hematoglobulin, is a molecular combination of 
hemoglobin and oxygen. The ability of hemoglobin to take up oxygen is a 
function of its iron content. When -this factor is calculated as 0.33-0.40 per 
cent., I atom of iron in the hemoglobin molecule corresponds to two atoms 
(one molecule) of oxygen. This combination is a loose one and hence the 
quantity taken up will depend upon the partial pressure of the oyxgen. This 
oxygen is set free when the oxygen pressure is reduced, thus giving rise to the 
characteristic property of oxygen transference. As Pflliger has shown, oxy- 
hemoglobin may, when it is gradually oxidized, act as an "ozone exciter" 
by the decomposition of neutral oxygen into the atomic form. It may also 
act as an "ozone transmitter" as in certain tests to be outlined later.^ 

A "dilute solution of oxyhemoglobin or of arterial blood shows a spectrum 
with two absorption bands between the Fraunhofer lines D and E. One 
band, a, is narrower, but darker and sharper and lies on the line D, the other, 
/?, is broader, less defined and less dark and lies at E. As the dilution becomes 
weaker the band /3 first disappears. By increased concentration the two 
bands become broader, the space between them smaller, and the blue and 
violet parts of the spectrum darkened. Other substances may give this same 
absorption spectrum, but oxyhemoglobin may be differentiated by its be- 
havior toward reducing agents, such as ammonium sulphid or Stokes' 
solution of ammoniacal ferrotartrate (see plate). 

Methemoglobin. 

This pigment is closely related to oxyhemoglobin, as it contains the same 
amount of oxygen and is isomeric with it. The oxygen is, however, not..ia 
loose combination and cannot, therefore, be utilized by the system. This 
coloring matter is formed by the spontaneous decomposition of blood, as 
observed in hemorrhagic transudates and cystic fluids, and occurs also in 
cases of posioning with potassium permanganate, potassium ferricyanid, 
chlorates, nitrites, nitrobenzol, acetanilid, antipyrin, turpentine, sulphonal, 
and arsenic, and in cases of cyanosis with diarrhea. 

According to Jaderholm and Bertin-Sans, the absorption spectrum of 
methemoglobin, in aqueous or slightly acidified solution, is similar to that of 

^ See McClendon, Jour. Biol. Chem., 191 5, XXI, 275. 



Absorption Spectra, 



^ 



PLATE XV. 





■ 




'4 


■ 


■ 1 


I^HH 


iSf^V 




_^B ft 1 






IH 


IH^^B 


[ ' ' 








^H ^^^^^^^^^H ^P^^iT^^T^^^^^^^^^^H 


It 1 s i 1 






^H 


IHE"1 


1^1 1 1 




^1 


I^^Hh 


BB ' '^IMV V^ 1 






■1 


^■^^H 


L i 1 



Oxyhaemoglobin. 



Haemoglobin. 



Carboxy- 
haemoglobin. 



Neutral Met- 
haemoglobin. 



Alkaline Met- 
haemoglobin. 



Alkali 
Haematin. 



ff^ROM HAWKS "pHyS/OLOG/CAL Cff£MISTRr"J 



Absorption Spectra. 



PLATE XVI. 



8 
9 
10 






*???? 


II 

12 

13 
14 


II 1 ■ 11 1 ■ 



fpROM HAWKS "PHrS/OLOG/CAL CH£M/STRr"J 



Reduced Alkali 
Haematin or 
Haemochromogen. 



Acid Haematin in 
ethereal solution. 



Acid Haemato- 
porphyrin. 



Alkaline 

Haematopor- 

phyrin. 



Urobilin or Hydro- 
bilirubin in acid 
solution. 



Urobilin or Hydro 
bilirubin -in alkaline 
solution after the 
addition of zinc 
chloride solution. 



Bilicyanin or 
Cholecyanin in 
alkaline solution. 



THE BLOOD 45 1 

acid hematin (see below), but is easily distinguished from the latter by the 
readiness with which it turns into that of hemoglobin on treatment with alkali 
and a reducing substance. Hematin, under the same conditions, gives the 
spectrum of an alkaline solution of hemochromogen^ (Hammarstein). Met- 
hemoglobin, in alkaline solution shows two absorption bands, which are like 
those of oxyhemoglobin, but differ from them in that the band nearer E is 
stronger than the one at D. A third fainter band may be observed, according 
to Hammarsten, lying between C and D. 

Carbon-monoxid Hemoglobin. 

This pigment is a molecular combination of one molecule of hemoglobin 
with one molecule of CO. This combination is stronger than those of hemo- 
globin and oxygen. The oxygen of hemoglobin is easily replaced, therefore, 
by CO and, in consequence, the tissues suffer for want of oxygen. This pig- 
ment imparts to the blood a bright cherry-red color both in the venous and 
arterial circulation. 

The most common cause of formation of the pigment is the inhalation of 
coal gas or of illuminating gas. The characteristic color in these cases may 
disappear after a few hours or it may persist for days, depending on the sever- 
ity of the case, being frequently found in the blood after death. Such blood 
shows an absorption spectrum similar to that of oxyhemoglobin, but the two 
bands lie more toward the violet end of the spectrum than in the case of oxy- 
hemoglobin. On the addition of a reducing agent to blood showing this 
spectrum, the absorption bands of carbon-monoxid hemoglobin are una- 
fected, while those of oxyhemoglobin are changed to those of hemoglobin. 
This spectroscopic test is not delicate as less than 20 per cent, of carbon- 
monoxid hemoglobin in the blood is difficultly, if at all, detected.^ 

Carbon-dioxid Hemoglobin. 

According to Bohr, hemoglobin forms three molecular combinations with 
CO2, in which products, a, /?, and 7, one gram of hemoglobin combines with 
1.5, 3, and 6 c.c. of CO2. The spectrum of these compounds is similar to 
that of reduced hemoglobin. If a large excess of CO2 be present the hemoglo- 
bin is decomposed and globin is precipitated. The. absorption band under 
these conditions is probably referable to the presence of free hemochromogen. 

Sulph-hemoglobin. 

This pigment, formed by the action of hydrogen sulphid upon hemoglobin, 
is found in the corpuscles and not free in the plasma. Sulph-hemoglobin- 
emia has been reported by several, especially by van der Bergh^ whose name 
is given to the condition known as ^'idopathic enterogenous cyanosis" and 
characterized by cyanosis, headache and marked constipation. Clarke^ has 
reported the first case in the United States. Spectroscopic examination of 

^ Halliburton and Rosenheim (Biochem. Jour., 1919, XIII, 195) suggest that the name 
of this pigment be changed to "reduced hematin." 

^ See Van Slyke and Salvesen, Jour. Biol. Chem., 1919, XL, 103, for a new method of 
determining CO in blood. Also Palmer, Ibid., 1918, XXXIII, 119. 

3 Deutsch. Arch. f. klin. Med., i9o5,LXXXIII, 86; Berl. klin. Wchnschr., 1906, XLIII, 7. 

4 Medical Record, 1909, LXXVI, 143; 1910, LXXVIII, 987. See, also, Wallis, Quart. 
Jour. Med., 1913, VII, 73; Jamieson, Ibid., 1919, XII, 81. 



452 



DIAGNOSTIC METHODS 



the diluted blood is essential to show the presence of this pigment. The 
spectrum shows 3 absorption bands, two similar to those of oxyhemoglobin 
and a third in the red not so near the C line as is the third band of methemo- 
globin. Addition of dilute ammonium sulphid destroys the third methemo- 
globin band, while it does not effect that due to sulph-hemoglobin. 

Decomposition Products of Blood Pigments. 

As stated previously, hemoglobin and oxyhemoglobin are proteins^ which 
are converted under the action of different physical and chemical means into 
a globulin-like protein, globin, and an iron-containing pigment, hemochromo- 
gen. From the table below, adapted from Webster and Koch,^ may be seen 
the relations of hemoglobin to its various derivatives and also to the bile 
pigments^ and to chlorophyll. 



Hemin 

ir. 




(C32H5oN,Fe03HCl) 
Hematin (C32H3i03N4Fe)-^Hematoporphyrin (CieH^gOaN^) 

/ li 

n/ Hemochromogen x PhyllopOTphyrin (CigHigNjO) 



Hemoglobin/ Hemochromogen 



Hemopyrrol (CgHigN)— Phyllocyanin<-Chlorophyll 



Urobilin (C32H40O7N,) 



Bilirubin {S:-^^^^.,0^± 



I^Hematoidiii 



Methemoglobin 




Biliverdin {^,^^^X)^ 



Hematin. 

This decomposition product is found in any situation in which oxyhemo- 
globin is destroyed; thus, in the digestive tract where it is formed by the ac- 
tion of gastric and pancreatic juice on oxyhemoglobin, in old extravsations, 
in the stools after hemorrhage, in urine after poisoning with arsenical 
compounds, and in the blood of persons poisoned with nitrobenzol and 

1 Laboratory Manual of Physiological Chemistry, Chicago, 1903. See Kiister, Ztsch. 
f. physiol. Chem., 1916, XCV, 152. 

2 See Austin and Pepper, Jour. Exper. Med., 191 5, XXII, 675; Krumbhaar, Musser and 
Peet, Ibid., 1916, XXIII, 87 and 97; Hooper and Whipple, Ibid., 137. 



THE BLOOD 453 

acetanilid.^ Hematin is a dark brown or blue-black amorphous powder, 
insoluble in water, dilute acids, alcohol, ether, and chloroform, but readily 
soluble in acidified alcohol or ether and in dilute alkalies. 

Arnold has shown that there are three modifications of this pigment, a 
neutral, an alkaline, and an acid hematin, each showing spectroscopic differ- 
ences. The neutral hematin has little importance chnically and will be passed 
in this discussion. Acid-hematin solutions show four absorption bands, one 
between C and D, a second broad but not clearly defined band between D and 
F, which divides under certain conditions into two narrower bands, a fourth 
band between D and E which is nearer D and is very weak. Usually only the 
band between C and D and the broad band between D and F can be seen (see 
plate). 2 

Solutions of alkaline hematin show one absorption band between C and 
D which reaches out to some extent between D and E. If the alkaline hema- 
tin solution be reduced with ammonium sulphid, the spectrum of hemo- 
chromogen is observed, which shows two characteristic absorption bands, 
one very sharp and dark between D and E and a second paler and broader 
band covering the E line. 

Hematin forms very characteristic compounds with hydrochloric, hydro- 
bromic, or hydriodic acids. With HCl hematin crystallizes with one mole- 
cule to form the compound hemin. The crystals are light or dark brown 
rhombic forms and are called, after their discoverer, Teichmann's crystals. 
Their formation is specific for blood, but the kind of blood cannot be deter- 
mined by their presence. As their detection is largely a medicolegal question, 
I will refer a discussion of the technic to the later section. 

Hematoporphyrin. 

When hematin is treated with concentrated sulphuric acid in presence of 
air, iron is split off leaving the pigment hematoporphyrin. If air be excluded 
the product yielded by such treatment is hematolin. 

This pigment is insoluble in water, but dissolves in alcohol, strong and 
weak alkalies, and in acids. It is isomeric with bilirubin, with which it is 
associated in the liver cells. In acid solution hematoporphyrin shows two 
absorption bands, one fainter and narrower between C and D and nearer 
D, the other darker, sharper and broader in the middle between D and E. In 
dilute alkaline solutions this pigment shows four absorption bands. A band 
between C and D, a second broader band surrounding D with the broadest 
part between D and E, a third between D and E nearly at E, and a fourth 
broad and dark band between E and F. Hematoporphyrin is of great impor- 
tance from the medicolegal standpoint as certain suspected stains may oe 
identified only by its spectrum. For this phase see the later sections. 

Hematoidin. 

This ruby-red or reddish-yellow pigment is derived from blood coloring 
matter and like hematoporphyrin is iron free. It is found in old blood-clots, 
in hemorrhagic exudates, in sputum, and in feces. It is more abundant 

1 Schumm (Ztschr. f. physiol. Chem., 191 2, LXXX, i) reports hematinemia in a case of 
acute chromium poisoning; Ibid., 1916, XCVII, 32. 

2 See Newcomer, Jour. Biol. Chem., 1910 XXXVII, 465. 



454 DIAGNOSTIC METHODS 

when the blood pigment is not much exposed to the action of living cells, as 
in the center of large extravasations and in hemorrhages into preformed cavi- 
ties of the body (Ziegler). Hematoidin isidentical with bilirubin and shows 
no absorption bands, but only a strong absorption of the violet to the green 
portion of the spectrum. 

Hemosiderin. 

This yellow, orange, or brown pigment is a derivative of hemoglobin and 
contains iron. Unlike hematoidin, it is found more particularly in extrava- 
sated blood which has been subjected to the action of living cells. After a 
time this pigment changes into one (probably hematoidin) which contains no 
iron. 

Malarial Pigment. 

The older view of Ewing^ and others that this yellowish-brown or black 
pigment was melanin seems to have been disproven by the later work of 
Brown^ who has shown that it is, probably, identical with hematin and is 
formed by the action of the malarial parasite (possibly of a proteolytic 
enzyme) upon hemoglobin. On spectroscopic examination of an alkaline 
alcoholic solution of this pigment, a single broad band is seen starting sharply 
at D and extending to the left, gradually shading into the red between C and 
D. The melanins, observed in the leucocytes in relapsing fever, melanotic 
sarcoma and Addison's disease, are quite distinct from this malarial pigment 
as shown by decolorizing and solubility reactions. 

Estimation of Hemoglobin. 

A very large number of methods have been introduced for the determina- 
tion of the blood coloring matter. Some of these are extremely accurate, but 
are too complicated for general . clinical purposes; others are less accurate, 
but are more applicable to our work. It may be said that, as a rule, the 
clinical instruments at our command will give comparative figures, providing 
their construction and their standardization is accurate. However, we find so 
much inaccuracy or even lack of proper standardization in some of these in- 
struments, that no absolute comparison may be attempted between the re- 
sults obtained by various workers using the same or different methods. 

Normal adult blood contains about 13.77 grams of hemoglobin per 100 
c.c. of blood, this figure being subject, however, to variations at different 
periods of life, so that these instruments which are standardized against nor- 
mal adult blood and which furnish the amount of hemoglobin in terms of 
percentage of such blood cannot be absolutely accurate no matter how per- 
fectly constructed or how accurately standardized, when they are used for the 
estimation of hemoglobin in the blood of a child or of an elderly patient. It 
is, therefore, much better practice to obtain the values in terms of the actual 
amount per 100 c.c. of blood. ^ It is well to recognize, as Tiirk points out, that 

1 Jour. Exper. Med., 1905, VI, 119. 

2 Arch. Int. Med., 1911, XIII, 290. 

•■'Williamson (Arch. Int. Med., 1916, XVIII, 505) has emphasized this point. His 
examination of 157 cases between the ages of 16 and 60 years shows that the hemoglobin, 
in the case of males, is 16.92 grams per 100 c.c, while with females it is 15.53. ^^ 227 cases 
between the ages of i day and 16 years shows, males, 16.20 grams; females, 16.38 grams. 



THE BLOOD 455 

we are working with possible errors and can obtain comparative results 
only when we use the same exactitude and care in each of our estimations. 
The errors are essentially constant, but we must remember that the arbitrary 
color standards change as time goes on, necessitating a restandardization of 
our instruments if accurate results are to follow. The writer has seen errors 
of 50 per cent, arise from the use of an old von Fleischl instrument and one 
of 25 per cent, from a faulty standardization of Sahli's solution. 

It must be insisted upon that we must not assume that a patient, who is 
pale and "anemic looking," is in reality anemic. Pallor depends not only 
upon the amount of pigment in the blood but also upon the delicacy and 
transparency of the skin and upon the superficial distribution and size of the 
blood-vessels. By remembering these points we may often save ourselves 
unnecessary chagrin on finding that the results of our determinations do not 
accord with the "anemic" expression of the patient. A simple puncture 
of the ear, allowing the blood to drop upon a clean linen towel (so-called 
" towel-test"), will many times set us right and prevent a diagnosis of anemia 
without further examination of the blood. Such examinationless diagnoses 
are unwarranted and inexcusable. 

Direct Methods of Estimation. 

These methods are, owing _to their complexity, not applicable to clinical 
work. The spectrophotometer of Hlifner is undoubtedly the very best and 
most accurate method, but I must refer to the original article for its descrip- 
tion. Likewise the colorimetric double pipet of Hoppe-Seyler and the meth- 
ods of Nebelthau and of Zangemeister must be neglected. 

Indirect Colorimetric Methods. 

These indirect methods employ a comparison of the blood solution with 
a second medium which approximates the blood in color. Two principles 
are possible in such methods. We may either have a fixed unchangeable 
medium of comparison, with which the blood is matched by constant dilution, 
or we may use a fixed blood solution of known strength and compare there- 
with the standard graduated color scale. Both of these principles have been 
used in such determinations, the variations being shown in the fact that in- 
struments for this purpose have been introduced by Gowers, Sahli, Hayem, 
Malassez, Henocque, Bizzozero, von Fleischl, Miescher, Haldane, Griitzner, 
Gartner, Dare, Oliver, and Tallqvist. 

It is evident, a priori, that these indirect methods must have greater 
errors than the direct. Two things are essential for any exactitude whatever. 
First, the medium of comparison must agree in the most complete manner 
with the various color tones which the blood shows at different percentage 
values of hemoglobin. Second, the standardization of the medium of com- 
parison must be extremely accurate. We must have no illusions regarding 
the exactness of the hemoglobin values obtained by these methods, as it is 
very difficult to construct two instruments of the same kind that will agree 
exactly. Certainly no two instruments of different make will agree, but 
comparative results sufficiently accurate for clinical purposes are obtained by 



456 



DIAGNOSTIC METHODS 



the use of the more reliable instruments. The personal equation in reading 
the color comparisons must be remembered, as some individuals show abnormal 
sensitiveness or lack of sensitiveness to shadings of red. It is an impossibihty 
in this book to give in detail all of the methods advanced for the estimation of 
hemoglobin. I select, therefore, those that have proven most reliable in my 
hands. 

Hemometer of Fleischl-Miescher. 

Up to recent times the most frequently used of the instruments for the 
estimation of hemoglobin was the old von Fleischl instrument. With the 
introduction of the Miescher modification, this original form has been or 
should be less often employed. We avoid, therefore, a discussion of the 
older instrument, referring to other works which have included it. 

This new apparatus, made by Reichert, under the direction of Miescher, 




Fig. 124— Hemometer of Fleischl-Miescher: R, Stage; T, miUed head, which moves 
the color scale; m, openmg m stage through which the instrument is read; M, mixing cell- 
D , cover glass; D, cap; PS, gypsum mirror from which light is reflected; mel dUutine 
pipet. ' ° 

is similar in general appearance to the old von Fleischl. It has the same 
stand and the same scale principle, although this latter is standardized differ- 
ently and graduated on a different basis. It differs, materially, in the method 
of measuring and diluting the blood, in the form of the comparison chamber, 
and in the meaning of the graduation of the scale (see cut and legend for its 
description). 

The Diluting Pipet. 

This is similar in construction to the pipet of the Thoma-Zeiss hemo- 
cytometer, its calibrations, however, being different. The marks are K, H, 



THE BLOOD 457 

and I. Above and below each of these main divisions are two marks each 
corresponding to Koo of the contents of the capillary tube. This device 
enables the worker to measure accurately the column of blood taken, in case 
he gets too little or too much blood in the tube. The relation of the capillary 
to the ampulla is such that blood, drawn to the mark i and diluted to the 
mark above the ampulla, receives a dilution of 200; if drawn to the mark % 
the dilution is 300; while the line J-^ furnishes a dilution of 400. The diluent 
used is Ho P^r cent, sodium carbonate solution. This dissolves the stromata 
of the red cells furnishing a clear solution. Occasionally the diluent becomes 
turbid after standing some time, and should be freshly made and should con- 
tain no bicarbonate. 

The taking of the blood and the mixing with a diluent is done with the 
same precautions mentioned under the method of making the blood count. 
In choosing the proper dilution, one should select that which will enable him 
to use the central portions of the graduated scale. A dilution of 400 is usually 
applicable except in cases in which marked anemia is suspected when dilutions 
of 300, 200, or even 100 should be made. In making this last dilution the 
erythrocytometer may be used. In doing accurate work, it is well to make a 
preliminary determination of hemoglobin in order to tell, the better, just what- 
dilution would be advisable. This may be done by the Tallqvist method 
described later. 

The Comparison Cells. 

With this instrument two cells for holding the blood and diluent are fur- 
nished. One of these has a depth of 15 mm., the other one of 12 mm. The 
former is the standard cell, the latter the control, giving % the value of 
the larger cell. Their external appearance is similar to the cell of the old 
Fleischl, but their capacity is less owing to their greater thickness. 

The dividing partition between the halves of the cells projects about 3^^ 
mm. above the borders, thus preventing any mixing of the fluids in the two 
portions. A grooved cover-glass is slid over the compartments without fear 
of mixing the fluids. If fluid is lost no error is introduced, as the dilution is 
uniform in the pipet and the depth of the chamber is definite. A diaphragm, 
with an opening 4 mm. wide, is placed over the cover-glass after this latter 
has been adjusted, the opening being so placed that its long axis is perpendicu- 
lar to that of the vertical partition. The field of comparison is thus limited 
to 4 mm., which corresponds to about 3° of the scale, affording a comparison 
of a single tint of the scale with the color of the diluted blood. 

Graduation of the Scale. 

The slide for comparison of color is tinted with Cassius' golden-purple, as 
in the old Fleischl instrument. The graduations of this color scale are made 
by comparison with standard solutions of hemoglobin and not with the ar- 
bitrary standard of blood of so-called normal individuals. The scale shows 
the same divisions as that of the Fleischl instrument, but their significance 
is different in the Miescher modification. Here one does not read directly 
the percentage of hemoglobin, but must obtain the corresponding value by 



45^ DIAGNOSTIC METHODS 

reference to a ''table of calibrations," which accompanies each instrument. 
While the figure thus obtained is in terms of actual percentage (grams per loo 
C.C.), it is quite different from the percentage figure given by the Fleischl 
instrument. 

Method. 

After testing the chamber by placing in one compartment the diluting 
fluid in order to see that none runs into the other, the blood is drawn into the 
pipet to the desired point and diluted to the mark, all precautions mentioned 
under Blood Counting being observed. Thoroughly mix the blood and dilu- 
ent by shaking and blow out the unmixed contents of the capillary tube. 
Fill one compartment of the 15 mm. cell with the diluted blood so that a con- 
vex meniscus appears above the border of the chamber, the other compart- 
ment being filled with the diluent or wdth distilled water. Adjust the grooved 
slide and cap, place the cell in the central opening of the stand, and adjust 
the light. This latter portion of the technic is of considerable moment, as this 
instruraent gives the best results when used in a dark room illuminated only 
by a small candle flame which is placed about 18 inches from the stand and to 
the side. In the absence of a dark room, a light-proof shield for the eyes may 
serve. This may readily be obtained by the use of a tube of stiff, dark paper, 
which fits over the comparison cell and shuts off the light of the candle from 
the field of vision. The observer should stand in such a position that he looks 
into the cell from the side and not from the front or back, the eyes being about 
one foot above the cell. In the comparison of the color tones the variations 
are much better seen by quick movements of the slide rather than by slow 
gradual changes. The eyes should be rested at short intervals to prevent 
fatigue and lack of sensitiveness to the different shadings of color. When 
the color is matched take the reading of the instrument, by observing what 
mark of the scale coincides with the notch on the edge of the opening above 
the scale, and control this reading by several duplicates. 

Remove the blood from the 15 mm. cell by means of the pipet and transfer 
it to one compartment of the 12 mm. chamber, tested as was the larger one. 
Adjust this chamber and make readings in the same way. These values 
should be only four-fifths of those obtained with the 15 mm. cell. This 
modification has the advantage of using different portions of the color scale 
and should give comparative figures. If there should be any variation, 
which should never be over i per cent., a correction may be made. 

Calculation of Results. 

This is possible only with the use of the ''table of calibrations," which con- 
tains the series of scale divisions and the absolute amount of hemoglobin in 
mg. per 1,000 c.c. of blood, corresponding to each division of the scale when 
the 15 mm. chamber is used. Thus, if the scale shows 56, we find the value 
corresponding to this to be 447 mg. of hemoglobin in 1,000 c.c. of diluted 
blood. As the dilution may have been 400, we would have, in 1,000 c.c. of 
undiluted blood, 400 x 447 = 178.8 grams. As we wish to know the amount 
in 100 c.c. we have merely to divide by 10, thus obtaining 17.88 grams of 



THE BLOOD 



459 



hemoglobin. If we wish to get the percentage figures, as read on the old 
Fleischl, we divide the figure obtained with the Miescher, in this case 17.88, 
by the amount of hemoglobin corresponding to the 100 division of the scale 
of the instrument used, in this case 14, and we obtain 127.7 per cent. 

This Miescher modification is our very best clinical apparatus for estima- 
tion of hemoglobin, giving results which are accurate within 0.2 to 0.5 per 
cent. It is open to the objections that it is bulky, expensive, and requires 
more time and practice for its use than is at the control of the busy practi- 
tioner. For hospital use, however, it is the instrument par excellence and 
should never be substituted by others. It has one disadvantage, that each 
instrument is standardized, not against a known hemoglobin solution, but 
against a so-called "normal" instru- 
ment, which has been properly stand- 
ardized. It is to be hoped that each 
instrument will, in the future, be 
properly calibrated and thus insure 
us against the possibility of errors 
arising from any change in the 
standardization of the "normal" 
instrument. 

Hemoglobinometer of Dare. 

The instrument, introduced by 
Dare, has the advantage of using un- 
diluted blood, and avoids any error 

consequent upon dilution. The prin- 

• 1 t i.-L.- ' J. 4. • £ ^^ Rj milled wheel; s, case inclosing the color 

ciple of this mstrument is as follows: ^isk; t, movabk wing, which is swung out- 
The color of undiluted blood is com- ward; u, telescoping camera; v, aperture 

pared by artificial Hght with that of a yf 'S'etadbawf cindl^ hXerY^, ^lot "through 
graduated glass scale colored with which the percentage of hemoglobin is read, 
golden-purple, the loo-point of which 

is standardized against a solution of 13.77 grams of hemoglobin in 100 c.c. 
of serum. For a description of the apparatus see cut. 

Method. 

Swing outward the movable screen, which serves as a cover for the case, 
adjust the camera tube, and fit the candle attachment in its place opposite 
the camera tube. The candle should be so adjusted that its upper end is 
flush with the top of the clips which hold it. If the wick be curved, it should 
be so turned that the intensity of the light is midway between the two aper- 
tures. See that the pipet, composed of the rectangular glass plates, is 
thoroughly cleansed and dry. The space between the plates is filled by 
applying the edge of the pipet to the side of a fairly large drop of blood. 
Adjust this pipet in its place and rotate the colored scale, by means of the 
milled screw, until the colors match. Hold the instrument steady to prevent 
the flickering of the flame as much as possible. No dark room is necessary, 
but it is advisable to point the instrument at some dark object and to avoid 




Fig. 125. — Hemoglobinometer of Dare: 



460 DIAGNOSTIC METHODS 

direct sunlight, as the shadings of color are not so easily matched by direct 
daylight. As soon as the colors are matched make the readings and check the 
results with several duplicates. This reading is observed on the left side of 
the case in the small open space, the line which coincides with the beveled 
edge of the opening representing the percentage of hemoglobin, on the basis of 
a value of 13.77 grams of hemoglobin per 100 c.c. as 100 per cent. It is, there- 
fore, easy to calculate the direct amount of hemoglobin in the blood examined. 
This instrument has the advantages that undiluted blood is used, that the 
scale of comparison is usually very accurately standardized, that it is con- 
venient, easy of manipulation, and rapid in giving results. Coagulation of 
the blood does not occur sufficiently soon to introduce an error, providing the 
reading is taken within a reasonable time. It is more convenient for general 




Fig. 126. — Method of filling the Dare blood pipet. {Da Costa.) 

use than is the Miescher, is less expensive, can be used in a light room, and 
gives results second only to those of the Miescher. The disadvantages of this 
instrument are that an occasional faulty standardization may introduce errors, 
it costs much more than some of the instruments to be described, and it is 
not a long-lived instrument unless care is taken in handling it. In the writer's 
laboratory this instrument has given great satisfaction and can be recom- 
mended for general use on the ground of its convenience and ready application 
to clinical work. 

Hemometer of Sahli. 
/ This instrument is a new modification of the older hemoglobinometer 
of Gowers, and has so many advantages over the older instrument that this 
latter will be passed over. A modification of the Gowers instrument intro- 
duced by Haldane is simple and reliable, but has the disadvantage that coal 
gas is used in converting the hemoglobin into CO hemoglobin, and consequently, 
is not of easy application for bedside work. 

As Sahli has pointed out, a colored fluid under examination should not 

be compared with a different substance similar in color, but with a solution 

of known strength of the same coloring matter. His modification of the 

Gowers method employs an instrument constructed on exactly the same lines, 

but using a different standard of comparison. This standard of comparison 

is an acid hematin solution^ in a concentration corresponding to a i per cent, 

^ Jacobson (Jour. A. M. A., 1919, LXXIII, 1282) proposes the use as a standard in this 
test, of a solution of gallic or tannic acid treated with concentrated sulphuric acid forming 



THE BLOOD 



461 



solution of normal blood. This solution is somewhat dark, as it is standard- 
ized against blood showing high hemoglobin value. As Sahli states, the stand- 
ard fluid, as furnished with his hemometer, corresponds to a blood which shows 
with the Miescher instrument at a dilution of 200 a reading of 109, or an ab- 
solute quantity of 17.2 grams of hemoglobin per 100 c.c. of blood. The fine 
particles of hemin, which are in suspension, may 
adhere to the glass, especially if the instrument lies 
unused for some time. This process changes the 
color of the standard to some extent, so that it is 
supposed that deterioration has occurred. This may 
be remedied by completely inverting the tube, without 
violent shaking, thus allowing the precipitate to dif- 
fuse uniformly. Occasionally one of these instru- 
ments is found to be improperly standardized, but 
this is rare. 

Method. 

This consists in diluting the blood with 10 times 
its volume of N/io normal hydrochloric acid. After 
a few seconds the fluid becomes dark brown from the 
formation of hematin hydrochlorid (hemin), which 
substance is not in solution, but in fine suspension. 
The blood is taken with the 20 cmm. pipet and 
blown into the graduated tube, which contains N/io 
normal HCl up to the mark 10. This acid may be 
accurately enough made by diluting 15 c.c. of concen- 
trated chemically pure HCl to 1,000 c.c. Shake the 
mixture of blood and acid and dilute with ordinary 
water as soon as a clear dark brown color is visible. 
Add water until the shade of the mixture corresponds Fig. 127.— Hemometer of 
exactly with that of the standard solution, when the 
percentage of hemoglobin may be read off. The 

water should be added very carefully from a dropping pipet, as the accuracy 
of the method depends upon adding the exact amount of fluid necessary. 
The comparison of colors may be made in any light, as the two solutions, 
being the same, will be similarly affected. 

This instrument is very conveniently gotten up, being mounted in a case, 
carrying a white-glass plate to reflect the light to better advantage. It is 
simple, inexpensive, and is accurate within 2 per cent. The author would 
recommend this instrument to the general worker above all others, with the 
possible exception of the Dare, whose advantages have been mentioned. 

Hemoglobinometer of Oliver. ♦ 

In this method the color of the blood in a definite dilution is compared, 

'rufgallic acid." See, also, Kuttner, Ibid., 1916, LXVI, 1370; Haessler and Newcomer, 
Arch. Int. Med., 1916, XVII, 806; Palmer (Jour. Biol. Chem., 1918, XXXIII, 119) advises 
the use of CO-hemoglobin as a standard. See, also, Cohen, Barnett and Smith, Ibid., 
1919, XXXIX, 489; Berman (Arch. Int. Med., 1919, XXIV, 553) proposes an acid hematin 
method, modifying the Sahli procedure. 




462 



DIAGNOSTIC METHODS 



by light reflected from a white surface, with a series of tinted glass standards. 
Such a method has the advantage that the color of the diluted blood is com- 
pared with one single tint at one time. The standard glass disks correspond 
accurately, as determined by the tintometer, to the specific color curve of 
progressive dilutions of normal blood. Two sets of standards are furnished, 




125.— Hemoglobinometer of Oliver. (Coplin.) 

one for use in daylight, the other by candlelight, the latter being preferable. 
Each set of 12 disks is mounted on plaster of Paris and enclosed in two 
wooden frames, six disks in each frame. They represent the color of solu- 
tions of hemoglobin with percentages from 10 to 120. To obtain interme- 
diate values, colored riders are used, each representing 2.5 and 5 per cent. 



THE BLOOD 



463 



of hemoglobin, when used with the disks from 70 to 120, but twice these 
values if used with the disks from 10 to 60. 

The blood is taken in a capillary pipet similar to that of von Fleischl, 
holding 5 cmm. of blood. The diluting chamber has a white background 
of plaster of Paris and, when filled with distilled water in which one pipetf ul 
of blood has been dissolved, yields a solution of i per cent. When filled the 
cell is covered with a blue-glass cover. 

Method. 

The pipet is filled with blood drawn by capillarity from a puncture of 
the ear or finger. Wash the blood from the pipet into the diluting chamber 
by means of water from a medicine dropper, fill the chamber with water, stir 
well with the handle of the pipet, and adjust the cover-glass. A small bubble 
of air should be included as an evidence that 
the chamber was not overfilled. 

Compare the color of this solution with 
that of the standard disks, using the light of a 
candle placed 3 to 4 inches away in such a 
manner that the light strikes the two cham- 
bers alike. It is well to use the camera tube to 
shield the eyes and enable one to compare 
the tints more accurately. If the color 
matches that of any of the disks, the percent- 
age is read directly from the case. If, how- 
ever, the color is intermediate between two 
disks, the riders must be superimposed on the 
disk of lower percentage, and a second com- 
parison made by using a corresponding plate 
of unstained glass as a rider on the diluting 
chamber. The adjustment of tints and riders 
must be continued until the colors match ex- 
actly. Naturally the variation will equal 2 
per cent, as the ordinary riders are equivalent 

to 23^ per cent, of hemoglobin. It is not necessary for this determination 
that the room be absolutely dark. 

This method is accurate within the limit mentioned (2.5 to 5 per cent.), 
but it is trying, time consuming, and does not equal in accuracy the methods 
of Dare and of Sahli. The instrument is inexpensive and the disks are prone 
to deteriorate. 

Tallqvist's Hemoglobinometer. 

Tallqvist has introduced a method for the estimation of hemoglobin 
which is so simple that certain writers have been led to recommend it above 
other methods. The principle of this method is essentially the same as Oliver's 
method, although the application is entirely different. Tallqvist compares 
the color of the undiluted blood with that of a series of lithographed standard 
tints, which range by differences of 10° from 10 to 100 per cent. These stand- 




FlG. 



129, — Tallqvist's hemoglo- 
binometer. 



464 DIAGNOSTIC METHODS 

ard tints were prepared by matching in water colors the tint of the blood of 
various patients (whose hemoglobin values had been determined with the 
von Fleischl instrument) when soaked into standard filter-paper. These tints 
were then lithographed and bound up with sheets of filter-paper, the com- 
bination making a very simple and convenient book which may be easily car- 
ried in the pocket. 

Method. 

Allow a fairly good-sized drop of blood to soak into a portion of the filter- 
paper by holding the edge of the sheet against the drop. Care should be taken 
to allow this to take place very slowly so that the color may be uniform through- 
out. If carefully done it will not be necessary to blot the stain, but some- 
times this is essential. As soon as the stain has lost its humid gloss compare 
its color with that of the lithographed scale. Do not allow the stain to become 
dry, as the color comparisons are disturbed by the coagulation. Hold the 
scale and the stained paper in such a way that the light (daylight only) is well 
reflected from the color scale. The percentage of hemoglobin is then read 
off directly by noticing the point of the scale with which the blood stain ex- 
actly corresponds. As this scale does not read closer than 10 per cent., the 
intermediate percentages must be judged by difference. Here personal 
factors are of great importance, some workers being so skilled as to detect 
small variations. 

This method can furnish, at best, only an approximate result and has 
nothing in common with the other methods discussed. It is to be advised 
only when some more reliable method is not at hand or when a rough estimate 
only is wanted. It is recommended by some writers as being as generally 
useful and accurate as any of the other methods, but the writer can see no 
advantage whatever in its use, as the results obtainable are, in his opinion, 
not reliable and are not as satisfactory to one who is not especially accustomed 
to such color comparisons. This test would better be limited to rough, pre- 
liminary, approximate determinations than to be used in the more careful 
estimations which clinical work requires. 

In the selection of a method for estimating the hemoglobin of the blood, 
the writer would advise the Fleischl-Miescher instrument for those who are in 
close touch with hospital or clinical laboratory facilities. For the general 
practitioner who must make his own determinations under varied circum- 
stances, the Dare or the Sahli instrument maybe unequivocally recommended, 
the latter having the advantage of cheapness along with accuracy and ease of 
manipulation. 

Variations m the Amount of Hemoglobin. 

The percentage values are misleading, as the hemoglobin varies with 
age and, to some extent, with sex.^ For this reason it is preferable to obtain 
the absolute amount of hemoglobin, which is done directly by the Miescher 
and, by a very simple calculation, with the Dare or Sahli instruments. In 
those methods which read in direct percentages one may readily calculate 

^ See Williamson, Jour. Am. Med. Assn., 1915, LXV, 302. 



THE BLOOD 465 

the absolute values per 100 c.c' of blood by multiplying the percentages ob- 
tained by 0.1377. Ii^ estimating the true figures for the hemoglobin in the 
blood of women, it is necessary to add one-eighth to the percentage values as 
read, as female blood runs from 123^ to 20 per cent, less in hemoglobin than 
does the blood of man; likewise for a child we should add one-seventh to the 
percentage. In this way we correct the readings of the instrument which 
is calibrated against adult male blood. It has been found by comparative 
tests, that the blood of rural residents reaches the normal standards much 
more frequently than does that of their city brothers, this being due, no 
doubt, to the purer fresh air which the country dweller enjoys. 

A relative diminution in the amount of hemoglobin, as determined by 
the above methods, is known as oligochromemia or as achroiocy themia. This 
condition is usually associated with a decrease in the number of erythrocytes 
(oligocythemia), but in cases of chlorosis the diminution of hemoglobin is an 
absolute reduction, each cell showing less hemoglobin than normally and no 
oligocythemia being, as a rule, present. In pernicious anemia, on the other 
hand, each cell may show an absolute increase in hemoglobin, although the 
percentage value is reduced owing to the marked oliogocythemia present 
at the same time. Factors bringing about conditions of hydremia lead to a 
transient oligochromemia, while factors acting as etiologic units in the pro- 
duction of anhydremia lead to a reverse condition of polychromemia. 

Oligochromemia is observed in chlorosis, pernicious anemia, leukemia, 
and secondary anemias, following chronic infections, hemorrhage, malignant 
growths, and some constitutional diseases. It is noted in chronic nephritis, 
chronic enteritis, and mineral poisonings, especially those with lead and 
mercury compounds. It has been stated that low hemoglobin values some- 
times observed in cases which are to have surgical intervention are contra- 
indications to operative treatment as the anesthetics themselves may bring 
on a condition of oligochromemia. While it is wise to watch with extreme 
care cases showing less than 50 per cent, hemoglobin, yet surgical operations 
have been successfully performed on cases with a more marked diminution 
of the hemoglobin. 

Color index. 

This term is used to indicate the amount of hemoglobin contained in each 
cell, as compared with the amount present when a normal number of erythro- 
cytes obtains. In other words, it is the quotient of the hemoglobin percent- 
age divided by the percentage of red cells. This latter factor is obtained by 
dividing the number of red cells, as found by the count, by the number reck- 
oned as normal, namely, 5,000,000. A very simple method of getting this 
percentage is to multiply the number of hundreds of thousands of red cells 
by two; thus if 2,650,000 reds were counted we obtain 53 as the percentage of 
red cells. Sahli recommends the use of the term "hemoglobin quotient" or 
"hemoglobin value" for this factor, but the expression color-index has become 
so general that it will be hard to replace. Moreover, the latter term seems to 
convey a more definite idea to most of us than would the substitutes suggested. 

The color-index is normally one; that is, a hemoglobin value of 100 per 



466 DIAGNOSTIC METHODS 

cent, is associated with a blood count of 5,000,000 red cells. We find, how- 
ever, in the different anemias that this figure varies markedly. In those 
anemias, in which the reds are diminished to the same relative extent as is 
the hemoglobin, the index remains normal; while in those cases in which the 
hemoglobin is markedly reduced without a coincident decrease in the reds, 
the value is necessarily less than one. This latter condition is observed es- 
pecially in chlorosis and in splenic anemia, while in the pernicious types of 
anemia we find the "diminution of the reds much greater, proportionally than 
that of the hemoglobin, a high color index consequently obtaining. In such 
cases the index may run from 1.02 to 1.9 as in one case observed by the writer. 

Such variations are the rule, but are not invariable. We may find the 
various anemias showing, at times, very unusual color indices. As this 
factor is intended to show only the relations of the hemoglobin to the cells 
it must not be taken as absolutely diagnostic, but rather as merely significant. 
It must be remembered, moreover, that this figure cannot be absolutely exact 
as it is based on a purely arbitrary number of red cells as the normal value and 
as the instruments for estimating hemoglobin are often improperly standard- 
ized. The results are, however, comparable and often yield valuable clinical 
information, if individual, racial, and seasonal variations in both the amount 
of hemoglobin and the number of red cells are taken into consideration.^ 
(C) Proteins of the Blood. 

From the point of view of physiological chemistry a discussion of the vari- 
ous protein bodies found in the blood embraces, necessarily, those of the 
intracellular fluid as well as those of the cellular elements. From the clinical 
standpoint, however, the discussion of this field is limited more or less to the 
proteins of the serum. I can, therefore, do little more than refer to the fact 
that the red cells contain, besides the hemoglobin which constitutes about 90 
per cent, of their organic matter, a nucleoprotein which shows properties 
resembling those of both the globulins and albumins. The proteins of the 
white cells are still little differentiated. Miescher found five different forms 
of protein, showing various solubility and coagulation relations. Besides 
these the leucocytes contain, as characteristic proteins, nucleins, which are 
compounds of the phosphoric acid-containing nucleinic acid with simple 
albumins. The metabolism of the nucleins is an important factor in various 
clinical conditions, but I must refer elsewhere for such discussions. Little is 
known regarding the chemical composition of the blood-plates. Lowit 
afl&rms that they are composed principally of globulin, while Lilienf eld asserts 
that their substance belongs to the class of nucleo-albumins. 

In the process of coagulation, fibrinogen, a protein of the plasma, is con- 
verted into fibrin through the influence of thrombin (fibrin ferment), whose 
chemical nature is not absolutely settled; it belongs probably to the class of 
nucleo-proteins, possessing, however, many of the characteristics of the globu- 
lins. Blood serum, of both physiologic and pathologic types, contains two 
protein bodies, serum globuHn and serum albumin. A third body, called by 
Chabrie albumon, has been assumed, but the researches of Drechsel and of 

1 See Meyer and Butterfield, Arch. Int. Med., 19 14, XIV, 94. 



THE BLOOD 467 

Brunner have shown that this body is not preformed in the serum, but arises 
from serum albumin and serum globulin during the process of coagulation. 

According to Hammarsten, normal human blood serum contains 7.62 per 
cent, of total protein, while Schmidt gives this figure as 8.26 per cent. The 
researches of Reiss, Strauss and Chajes, and more recently of Engel have 
shown that the refraction coefficient of serum, in health, is between 1.3487 
and 1.35 1 7, corresponding to a percentage of 7.74 to 9.13 of protein. The 
introduction of the refraction coefficient into the study of the serum and 
plasma has furnished a method of clinically studying hydremia as well as one 
by which the water content and serum proteins may be easily estimated.^ 
These factors are of great importance in the study of the various anemias, as 
we know that the serum or plasma is of much more importance, from the 
pathologic standpoint, than are the variations in the celllular structures, 
which are simply manifestations of profound changes in the liquid portions 
of the blood. 

In this connection we must distinguish between an increase in the proteins 
of the blood (hyperalbuminosis) and a decrease in their amount (hypalbumi- 
nosis) . The former is observed whenever water is more rapidly withdrawn from 
the system, and so from the blood, than it can be supplied. Such conditions 
are furnished by marked diarrhea, cholera, profuse perspiration, and polyuria 
without extra intake of fluid. This increase in protein content is only 
transient and is a result of mere concentration of the blood, the proteins 
passing out in relatively less amount than does the water. If the above con- 
ditions are associated with true exudation, then, of course, a hypalbuminosis 
of transient duration will replace the hyperalbuminosis. This decrease in the 
amount of total proteins is observed whenever direct loss of protein from the 
blood occurs. Thus Becquerel and Rodier observed a diminution of the pro- 
teins in Bright's disease, cardiac edema, and puerperal fever. Hoppe-Seyler 
noted a loss in melanosarcoma, while Schmidt, von Jaksch, Panum and 
Limbeck, and Pick have reported such a condition in severe anemias and 
leukemias. In most severe infections, as Ewing states, the proteins are but 
slightly reduced. Along with hypalbuminosis we often observe a hydremia 
which may be referable to a direct absorption of fluid from the tissue under the 
influence of a hypertonic state of the blood. 

In contrast to the constancy of the total protein values of the serum, we 
find the relationship between the albumin and globulin markedly disturbed 
at times. These two bodies are in reality not definite chemical compounds, 
but are separable, each into two distinct substances with different solubilities 
and precipitation constants. This fact may have great importance as further 
study is made, inasmuch as Pick has shown that pseudoglobulin has asso- 
ciated with it the antitoxins of diphtheria and of tetanus. ^ The normal 

^ See Schorer, Cor.-Bl. f. schweiz. Aerzte., 1913, XLIII, 1523; also, Lowy, Deutsch. Arch. 
f. klin. Med., 1914, CXV, 318; Robertson, Jour. Biol. Chem., 1915, XXII, 233; Tranter and 
Rowe, Jour. Am. Med. Assn., 1915, LXV, 1433; Reiss, Deutsch. Arch. f. klin. Med., 1915, 
CXVII, 175; Rakuzin, Jour. Russ. Phys.-Chem. Soc, 1916, XL VIII, 1251; Rowe, Arch. 
Int. Med., 191 7, XIX, 354. 

2 The coagulative elements of blood serum appear to be closely associated with the 
euglobulin fraction. See Hess, Jour. Exper. Med., 1916, XXIV, 701; Berg, Jour. Agricul. 
Res., 191 7, VIII, 449. 



468 DIAGNOSTIC METHODS 

amount of total protein being taken as 7.62 per cent., Hammarsten has shown 
that this percentage is made up of 3.10 per cent, of serum globulin and 4.52 
per cent, of serum albumin, the relationship of globulin to albumin being as 
1:1.5. This ratio is variable, running, according to Limbeck and Pick, globu- 
lin 16.9 to 38.3 per cent, of total protein, albumin 61.7 to 83.1 per cent, of the 
total albuminous content. Such a wide variation makes it difficult to estab- 
lish any absolute relations in disease. Erben has shown that the albumin 
remains about normal in pernicious anemia, while the globulin is markedly 
reduced. The researches of Estelle, Hoffmann, Halliburton, and Mya and 
Viglezio show marked pathological variations not only in the total protein 

n 1 a . . ,, albumin ^, , 

content, but m the so-called protem quotient, ~r^r~p — The latter 

authors conclude among other deductions that the relation of the proteins is 
greatly changed in disease, especially in conditions associated with transuda- 
tion and exudation, in the sense that the globulins^ are' increased while the 
albumins are diminished. In such states globulin is seen to be relatively more 
resistant and less diffusible than is albumin (Gottschalk). That this view is 
not uniformly applicable is noted from the work of Freund who observed in 
the serum of nephritis a relationship of i : 11.3 between the globulin and albu- 
min, instead of the normal 1:1.5. Ducceschi has reported an interesting 
observation on this point. He found that, during the period preceding the 
convulsions which follow thyroidectomy, a percentage increase of albumin as 
regards globulins obtains, while during the convulsions the reverse conditions 
are present. 

In this discussion I have taken no account of the total protein of the whole 
blood. In its determination we include not only the albumin and globulin of 
the serum, but also the hemoglobin of the red cells, the nucleo-proteins of the 
white cells, and the fibrinogen of the plasma. Traces of albumose and pep- 
tone( ?) have been found in pathological conditions, the former possibly in nor- 
mal states, while still other nitrogenous constituents are commonly deter- 
mined with the proteins. 2 The total protein of the blood has been given by 
von Jaksch as 22.62 per cent., while Limbeck states a percentage of 25. So 
much depends upon the method adopted for its estimation and so much on 
the physiological state of the blood that comparative figures are difficultly ob- 
tainable from the literature. Regarding the determination of the total protein 
and of the globulin and albumin, the reader is referred to the section on Urine. 

Regarding the question of the presence of peptone in the blood, as reported 
by von Jaksch, Freund and Obermayer, and Ludwig, much depends on the 
proper differentiation of the substance found. So much has been called 
peptone that is, in reality, albumose, that we are uncertain whether peptone 
was present or, if it were, whether it was not formed in the processes used 



^ See Epstein, Jour. Exper. Med., 191 2, XVI, 719; Hurwitz and Meyer, Ibid., 1916, 
XXIV, 515; Rowe, Jour. Lab. and Clin. Med., 1916, I, 485; Arch. Int. Med., 1916, XVIII, 
455; Ibid., 191 7, XIX, 499; Hurwitz and Whipple, Jour. Exp. Med., 1917, XXV, 231; 
Hanson, Jour. Immunol., 1918, III, 139. 

2 See Folin and Denis for a discussion of protein metabolism as shown by blood examina- 
tions (Jour. Biol. Chem., 1912, XI, 87 and 161; Ibid., 1912, XII, 141; Ibid., 1913, XIV, 29). 



THE BLOOD 469 

or was not a postmortem product. Devoto and Wagner could not confirm 
the finding of true peptone when blood from the living subject was examined. 
It is rather strange that peptone, which reduces the coagulability of blood 
when added in small amounts, should not exert this power in the vessels 
during life were it really present. The fact, however, that peptone and 
albumose have both been repeatedly found in the urine in various /conditions 
by competent observers points totheprobabiHty of the existence of these prod- 
ucts in the blood, although they may be combined in such a way as not to be 
capable of easy detection. Further elaboration of our methods of detection 
and differentiation may clear up a much disputed field. Bywaters believes 
the so-called albumose of the blood to be identical with sero-mucoid. 

(D) Other Nitrogenous Constituents. 

Other nitrogenous bodies than those already discussed are found in the 
blood in normal and, in varying amounts, in abnormal conditions. Owing to 
the introduction of more exact methods of study, especially of the micro- 
chemical methods of Folin and his coworkers, extensive investigation of the 
non-protein nitrogenous elements of the blood has been undertaken, to the 
end that variations in the amount of total non-protein nitrogen as well as of 
the different specific nitrogenous bodies are becoming of great importance in 
both diagnosis and prognosis. It is unquestionably true that a study of the 
relationship between the urinary nitrogen factors and those of the blood gives 
a much more certain index of functional perversion as well as of the extent of 
this abnormality than does the investigation of either group of factors alone. 

Total Nitrogen. 

In the determination of the total nitrogen of the blood, one includes the 
nitrogen referable to the various protein elements as well as that derived from 
the different non-protein nitrogenous bodies. The method adopted in this in- 
vestigation is the usual Kjeldahl method discussed on page 222, using 5 c.c. of 
blood to which a little potassium oxalate is added as withdrawn in order to 
prevent coagulation and to insure exact measurement of the volume desired. 

This factor, in itself, has very little importance. Von Jaksch gives the 
total nitrogen of normal blood serum as 1.37 per cent., while that of the whole 
blood he finds is 3.62 per cent. Variations in these figures are no doubt fre- 
quent and depend upon changes in the amount of the many different elements 
making up the total blood nitrogen.^ 

Total Non-protein Nitrogen. 

While the total nitrogen referable to the protein elements of the blood 
shows a practically constant value, we find the non-protein elements subject 
to considerable variation. Although the amounts of the various non-protein 
nitrogen factors are relatively small as compared with those of the protein 
constituents, yet we find the percentage and absolute relationships between 
the total non- protein nitrogen, on the one hand, and the various elements of 
which it is composed, on the other, showing such distinct variations from the 

1 See Grafe, Deutsch. Arch. f. klin. Med., 1915, CXVIII, 149; Mosenthal and Richards, 
Arch. Int. Med., 1916, XVII, 329; Buell, Jour. Biol. Chem., 1919, XL, 63. 



470 DIAGNOSTIC METHODS 

normal in certain pathological conditions that diagnostic and prognostic 
deductions may be made with increased certainty. For this reason a close 
study of these factors is of great importance. 
Method of Folin and Wu. 

This method^ is a modification of the earlier methods of Folin and Denis. ^ 
The chief reason for the introduction of this modification is the formulation 
of a system of blood analysis, which will permit of the determination of a 
maximum number of nitrogenous constituents with one precipitation of pro- 
tein material. The reagents used for this purpose have been many, most of 
them having certain faults. Thus Folin and Denis originally used methyl 
alcohol followed by an alcoholic solution of zinc chlorid; later, these workers 
employed a solution of meta-phosphoric acid; Greenwald^ first advocated 
the use of 2.5 per cent, solution of trichloracetic acid followed by kaoHn and 
later modified his method by using a 5 per cent, solution of this acid as the 
precipitant; Gettler and Baker^ use mercuric chlorid, while Welker and Falls^ 
consider alumina cream as especially valuable for serum; recently Fischer^ 
has advised the use of uranium acetate. While the use of tungstic acid, as 
employed in this test, does not yield any better results, as far as the precipi- 
tation of the blood protein is concerned, than do the trichloracetic acid of 
Green wald or the meta-phosphoric acid of Folin and Denis, yet this proced- 
ure admits of the determination of practically all the non-protein nitrogenous 
elements of the blood and is, therefore, recommeded here as the precipitant 
of choice. 

Obtaining the Blood. 

To the tip of a perfectly clean, dry serological pipet, attach the usual 
serological needle by means of a short piece of pure gum tubing. Drop 
a small pinch of powdered potassium oxalate (citrate should be avoided in ^ 
this test, except in minimal amounts) into the upper end of the pipet and 
allow it to run down into the tip (20 mg. of the oxalate is sufficient for 10 
c.c. of blood, more interferes with the later uric acid determinations) . Con- 
nect the upper end of the pipet with a rubber suction tube, which is, in turn, 
connected with a short glass mouthpiece. Insert the needle into the median 
basilic vein and, by suction, fill the syringe with the blood. It is not neces- 
sary that any definite amount be drawn, as the method is adaptable to any 
obtainable amount of blood. It is wise, however, to draw at least 10 c.c. if 
possible, which will permit of duplicate determinations if desired. The 
blood may, then, be transferred to a clean test tube and kept in the ice box 
if the determination is not to be made at once. 
Precipitation of Protein. 

Transfer a measured amount of blood into a flask having a capacity of 15 
or 20 times that of the volume taken. It is not necessary that volumetric 

1 Jour. Biol. Chem., 1919, XXXVIII, 81. 

2 Ibid., 1912, XI, 527; Ibid., 1916, XXVI, 491. 

3 Ibid., 191S, XXI, 61; Ibid., 1918, XXXIV, 97. 
^ Ibid., 1916, XXV, 210. 

' Ibid., 567. 

^ Ztschr. f. physiol. Chem., 1918, CII, 266. 



THE BLOOD ' 47 1 

flasks be used. For the measurement of the blood, special pipets graduated 
to 15 c.c. are recommended, but the usual Mohr's 10 c.c. graduated pipets 
are very satisfactory. Dilute the blood with 7 volumes of water and mix. 
With an appropriate pipet add i volume of 10 per cent, solution of sodium 
tungstate (Na2W042H20) and mix. With another suitable pipet add to 
the contents in the flask (with shaking) i volume of % normal sulphuric 
acid. Close the mouth of the flask with a rubber stopper and give a few 
vigorous shakes. When the blood is properly coagulated, the color of the 
coagulum gradually changes from pink to dark brown. If this change does 
not occur, the coagulation is incomplete, due to too much oxalate or citrate. 
In such an emergency, add 2N. sulphuric acid, drop by drop, shaking vigor- 
ously after each addition and allowing the mixture to stand for a few minutes 
before adding more. Pour the mixture on a filter large enough to hold the 
entire contents of the flask and cover with a watch-glass. If this filtration 
is begun by pouring the first few c.c. of the mixture down the double portion 
of the filter paper and withholding the remainder till the whole filter has- 
been wet, the filtrates are almost invariably as clear as water from the 
first drop. If the filtrate is not perfectly clear, the first few c.c. may have to 
be refiltered. As Folin and Wu state, this precipitation is so simple that no 
one need go astray, provided that the sodium tungstate and the J^ normal 
sulphuric acid are correct. The quality of the tungstate may not be what it 
should be, owing to a large percentage of carbonate. This carbonate content 
may be determined as follows: To 10 c.c. of the 10 per cent, solution, add 
one drop of phenolphthalein solution and titrate with o.i N. hydrochloric 
acid. Each c.c. of the HCl corresponds to 1.06 per cent, of sodium carbon- 
ate. The amount of acid required for the titration should not exceed 0.4 
c.c. The amount of sulphuric acid, used in the test, is intended to set free 
the whole of the tungstic acid with about 10 per cent, excess (and to neu- 
tralize the carbonate usually present in commercial tungstates). A greater 
excess of sulphuric acid must not be used, as a large part of the uric acid 
will be lost by such procedure. If the filtrate, obtained as above, -is to 
be kept for any length of time, one or two drops of toluol or xylol should be 
added to it. 

Technic. 
Introduce 5 c.c. of the protein-free blood filtrate (corresponding to 0.5 
c.c. of blood) into a clean (washed with water and alcohol) and dry 75 c.c. 
Pyrex test-tube, which is graduated at 35 and at 50 c.c. Add i c.c. of the 
sulphuric-phosphoric acid digestion mixture. ^ Add a dry quartz pebble 
or a dry glass bead and boil vigorously over a microburner until the charac- 

^ This digestion mixture is made up as follows: Mix 300 c.c. of phosphoric acid syrup 
(about 85% H3PO4) with 100 c.c. of concentrated sulphuric acid. Transfer to a tall cy- 
linder, cover well to exclude the absorption of ammonia, and set aside for sedimentation 
of calcium sulphate. This is very slow, but in the course of a week or so the top part is 
clear and may be drawn off by means, of a pipet. (It is not absolutely necessary that 
the calcium be removed, but it is probably safer to have it done). To 100 c.c. of the clear 
acid add 10 c.c. of 6 per cent, copper sulphate solution and 100 c.c. of water. 2 c.c. of this 
solution are equivalent to i c.c. of the acid mixture described on page 226. 



472 DIAGNOSTIC METHODS 

teristic dense acid fumes begin to fill the tube. This is usually accomplished 
in from 3 to 7 minutes. When the fumes are unmistakable, cut down the 
size of the flame so that the contents of the tube are just visibly boiling, and 
close the mouth of the tube with a watch-glass or a small Erlenmeyer flask. 
Continue the heating very gently for 2 minutes from the time the fumes 
begin to be unmistakable, even if the solution has become clear and colorless 
at the end of 20 to 30 seconds. If the oxidations are not visibly finished at 
the end of the 2 minutes, the heating must be continued until the solution is 
nearly colorless. Allow the contents to cool for 70 to 90 seconds and then 
add 15 to 25 c.c. of water. Cool further, approximately to room temperature, 
and add water to the 35 c.c. mark. Add, preferably with a pipet, 15 c.c. of 
Nessler's solution.^ Insert a clean rubber stopper and mix. If the solution 
is turbid, centrifuge a portion before making the color comparison with 
the standard solution of ammonium sulphate (see page 226), The standard 
most commonly required in this test is 0.3 mg. of N, which amount should be 
placed in a 100 c.c. flask. Add to it 2 c.c. of the digestion mixture, about 
50 c.c. of water, and 30 c.c. of Nessler solution. Fill to the mark and mix. 
The unknown and the standard should be Nesslerized at approximately 
the same time. If the standard, in the colorimeter, is set at 20 mm. for the 
color comparison, then 20 divided by the reading of the unknown 9,nd 
multiplied by 30 gives the non-protein nitrogen in mg. per 100 c.c. of blood. ^ 

Amount of Non-protein Nitrogen. 

This non-protein nitrogen of the blood has been given various names in 
times past, among which we find "incoagulable nitrogen;" "extractive nitro- 
gen;" "filtrate nitrogen;" " non-dialyzable nitrogen;" "rest," "waste," or 
" retention nitrogen." Many figures have been given by various workers for 
this factor, but these depend, to a large extent, on the method employed for 
its determination as well as upon the state of digestion at the time the blood is 
drawn for examination. It has been clearly demonstrated that the "non- 
protein nitrogen of the blood does rise and sink like a tide with reference to 
absorption from the digestive tract." This "digestive rise" is, of course, not 
a very great one, but it is sufficient to necessitate a slightly variable figure for 

^ This Xessler solution may be prepared as directed on page 226. As the mercuric 
iodid, no^Y purchasable on the market, may contain insoluble impurities, which make it 
difficult to obtain a clear solution on the addition of potassium iodid, the following method 
of preparing this reagent is recommended: Transfer 150 grams of potassium iodid and no 
grams of iodin to a 500 c.c. Florence flask; add 100 c.c. of water and an excess of metallic 
mercury (140 to 150 grams). Shake the flask continuously and vigorously for 7 to 15 
minutes or until the dissolved iodin has nearly disappeared. The solution becomes quite 
hot. When the red iodin solution has begun to become visibly pale, though still red, cool 
in running water and continue the shaking until the reddish color has been replaced by the 
greenish color of the double iodid of mercury and potassium. This operation requires 
about 15 minutes. Now separate the solution from the surplus mercury by decantation 
and washing with liberal quantities of distilled water. Dilute the solution and washings 
to a volume of 2 liters. If the cooling is begun in time, the resulting reagent is clear enough 
for immediate dilution with alkali. Introduce into a large bottle 3500 c.c. of 10 per cent, 
sodium hydrate solution, add 750 c.c. of the double iodid solution and 750 c.c. of distilled 
water, giving 5 liters of Nessler's solution. 

^ See Bock, Jour. Biol. Chem., 1916, XXVIII, 357; Grigant and Guerin, C. R., soc. 
bio]., Paris, 1918, LXXXI, 1139; Langstroth, Jour. Biol. Chem., 1918, XXXVI, 377; 
Peters, Ibid., 1919, XXXIX, 285. 



THE BLOOD 473 

the normal value of non-protein nitrogen of the blood. Folin and Denis con- 
sider the normal value to be between 22 and 26 mg. per 100 c.c. of blood. 
During digestion the rise is about 4 mg., so that we may consider a normal 
non-protein figure to be below 30 mg. per 100 c.c. of blood. 

As the kidney is the great regulator of the composition of the blood, main- 
taining a practically constant level of non-protein nitrogen, it is in disorders of 
this organ, especially, that most is to be expected from a study of the variations 
in the non-protein nitrogen of the blood. In the interpretation of an increase 
in the non-protein nitrogen in nephritis Mosenthal considers that four factors 
must be regarded: (i) retention of nitrogen by an insufficient kidney; (2I in- 
spissation of blood due to loss of water; (3) increase of protein catabolism; 
and (4) the chemical combination in which the non-protein nitrogen exists 
in the blood. In cases of high fever, -associated with marked toxogenic de- 
composition of protein, it might be rational to suppose that these de- 
composition products would find their way into the blood current and increase 
the non-protein nitrogen of the blood. As a matter of fact we do find in 
pneumonia a considerable increase, as a rule, reaching its maximum toward 
the crisis but bearing no relation to the time at which resolutipn takes place. 
In very few of the other fevers, however, has a rise of any importance been noted. 

Numerous workers have shown that, in the majority of cases, the non- 
protein nitrogen increases with an increasingly severe nephritis. In many 
cases of nephritis, however, the non-protein nitrogen as well as the urea fall 
within normal limits. It is probably true that, in both chronic interstitial and 
chronic diffuse nephritis, cases without symptoms of uremia show normal or 
only moderately elevated values. In cases showing considerable hyperten- 
sion, the non-protein nitrogen may be much increased. In cases tending 
toward uremia or showing actual uremia, the values are markedly increased, 
reaching in some cases as high as 350 mg. per 100 c.c. Amounts of 100 mg. or 
over per 100 c.c. are rarely seen in conditions other than uremia, so that this 
factor assumes great importance in diagnosis. Further, the prognostic 
value of this examination is shown in the fact that patients with high non- 
protein nitrogen do not, as a rule, survive for a very long period. Another 
valuable point in the study of this factor is that it furnishes a guide to the 
proper diet to be allowed nephritics, as cases with high retention require 
restriction of protein. In chronic passive congestion of the kidneys there is 
little or no retention, while in chronic lead poisoning, which is so often asso- 
ciated with granular kidneys, considerable retention may be observed, but 
the figure never reaches that shown in uremic conditions. In the eclampsias 
of pregnancy we seldom find a marked increase in the non-protein nitrogen, at 
least so far as reported cases are to be considered. This slight retention, in 
obscure pregnancy, may serve as a point in differential diagnosis from uremia.^ 

1 See Folin and Farmer, Jour. Biol. Chem., 1912, XI, 493; Folin and Denis, Ibid., 87, 161, 
503 and 527; Ibid., 1912, XII, 141 and 253; Folin and Lyman, Ibid., 259;Lowy, Ztschr. f. 
physiol. Chem., 1912, LXXIX, 349; Folin and Denis, Jour. Biol. Chem., 1913, XIV, 29; 
Ibid., 1913, XVII, 487 and 493; Michaud, Cor.-Bl. f. Schweiz. Aerzte, 1913, XLIII, 1474; 
Farr and Austin, Jour. Exper. Med., 1913, XVIII, 228; Karsner and Denis, Ibid., 1914, XIX, 
259; Folin, Denis and Seymour, Arch. Int. Med., 1914, XIII, 224; Agnew, Ibid., 485; 
Frothingham and Smillie, Ibid., 1914, XIV, 541; Tileston and Comfort, Ibid., 620; Mosen- 



474 DIAGNOSTIC METHODS 

Urea. f 

This is, of course, the most important of the single elements which make 
up the total non-protein nitrogen of the blood. Its importance Hes in the fact 
that its accumulation in the blood runs parallel|to that of the total non- 
protein nitrogen and, hence, is of much diagnostic andjprognostic value. 

Method of Van Slyke and Cullen. 

This method is an adaptation of the same method discussed in the sec- 
tion on urine. Three c.c. of fresh blood or other body fluid are measured 
with an accurate pipet and run into a loo c.c. heavy Jena test-tube contain- 
ing I c.c. of 3 per cent, potassium citrate solution to prevent clotting. 0.5 
c.c. of urease solution (prepared by the method of Van Slyke and Cullen, see 
Urine, page 234) and two or three drops of caprylic alcohol are added. Allow 
this mixture to stand 10 minutes and then add 5 c.c. of saturated potassium 
carbonate solution. Drive off the generated ammonia by means of an air 
current into 25 c.c. of N/ioo hydrochloric acid. Titrate back the excess 
of acid with N/ioo sodium hydrate solution, using methyl red as an indi- 
cator. Each c.c. of N/ioo acid neutralized indicates 0.00014 gram nitrogen. 
As 3 c.c. of blood were used in this process, we find that each c.c. of N/ioo 
acid neutralized is equivalent to 0.00467 gram of nitrogen referable to urea 
or to o.oi gram of urea per 100 c.c. of blood. As fresh blood contains so little 
ammonia, as such, it may be disregarded in this process. 

Method of Folin and Wu. 

This method^ is a part of the system of blood analysis arranged by these 
workers and is, in reality, a modification of the method of Van Slyke and 
Cullen just discussed. It consists of the decomposition of the urea in the 
blood filtrate to ammonia by the use of jack bean urease in the presence of a 

thai, Ibid., 844; Gulick, Jour, Biol. Chem., 1914, XVIII, 541; McLean and Selling, Ibid., 

1914, XIX, 31; Pribram, Zentralbl, f, inn. Med., 1914, XXXV, 153; Hertz, Wien. klin. 
Wchnschr., 1914, XXVII, 323; Austin and Miller, Jour. Am. Med. Assn., 1914, LXIII, 
944; Farr and Krumbhaar, Ibid., 2214; Farr and Williams, Am. Jour. Obs., 1914, LXX, 
614; Am. Jour. Med. Sc, 1914, CXLVII, 556; Frothingham, Ibid., 1915, CXLIX, 808; 
Plass, Am. Jour. Obs., 1915, LXXI, 608; Foster, Arch. Int. Med., 1915, XV, 356; Fitz, 
Ibid., 524; Hopkins and Jonas, Ibid., 964; Schultz and Pettibone, Am. Jour. Dis. Child., 

191 5, X, 206; Tileston and Corofort, Ibid., 278; Hohlweg, Mitt. a. d. Grenzgeb. d. Med. u. 
Chir., 1915, XXVIII, 459; Med. EUin., 1915, XI, 331; Bock and Benedict, Jour. Biol. 
Chem., 1915, XX, 47; Myers and Fine, Ibid., 391; Greenwald, Ibid., 1915, XXI, 61; 
Harding and Warneford, Ibid., 69; Folin, Ibid., 195; Taylor and Hulton, Ibid., 1915, XXII, 
63; Taylor and Lewis, Ibid., 71; Pepper and Austin, Ibid., 81; Folin and Denis, Ibid., 

1915, XXII, 321; Woods, Arch. Int. Med., .1915, XVI, 577; Christian, Frothingham, 
G'Hare and Woods, Am. Jour. Med. Sc, 1915, CL, 655; Wolf and Gutmann, Deutsch. 
Arch. f. klin. Med., 1915, CXVIII, 174; Bang, Biochem. Ztschr., 1916, LXXII, 104, 119, 
129, 139, and 146; Pozzilli, Policlinico, 1916, XXIII, 33; Scheel, Ugesk. f. Laeger, 1916, 
LXXVIII, 493 and 541; Slemons and Morris, Bull. Johns Hopk.^Hosp., 1916, XXVII, 
343; Cooke, Rodenbaugh, and Whipple, Jour. Exper. Med., i9i6,'^XXIII, 717; Karsner, 
Jour. Lab. and Clin. Med., 1916, I, 910; Leopold and Bernhard, Am. Jour. Dis. Child., 

1916, XI, 432; Rosenberg, Berl. klin. Wchnschr., 1916, LIII, 1314; Minsk and Sauer, Am. 
Jour. Dis. Child., 191 7, XIII, 397; Mosenthal and Hiller, Jour. Urol., 191 7, I, 75; Whipple 
and Van Slyke, Jour. Exper. Med., 1918, XXVIII, 213; Feigl, Arch. f. exp. Path, und 
Pharm., 1919, LXXXIII, 299, 317, and 335; Brun, Acta Med. Scand., 1919, LII, 367; 
Slemons, Canad. Med. Quart., 1919, II, 447. 

^ Jour. Biol. Chem., 1919, XXXVIII, 81; see, also, Folin and Denis, Ibid., 1912, XI, 
527; Ibid., 1916, XXVI, 505; Landolph, Sem. Med., 1916, XXIII, 337; Hulton-Frankel, 
Jour. Lab. and Clin. Med., 1918, III, 548. 



THE BLOOD 475 

new phosphate solution and the distillation or aeration of the ammonia into 
a known solution of dilute acid. 

Technic. 

Transfer 5 c.c. of the tungstic acid blood filtrate (previously discussed 
under Non-protein Nitrogen on page 470) to a clean and dry Pyrex ignition 
tube (capacity about 75 c.c.)-^ Add to the blood filtrate two drops of the 
sodium pyrophosphate solution, whose composition is given below. ^ Then 
add 0.5 to I c.c. of the jack bean urease solution,^ and immerse the test 
tube in a beaker of warm water and leave it there for 5 minutes. The tem- 
perature of the water is not especially important, but should not exceed 55° 
C. If one prefers not to use the hot water bath, room temperature may be 
employed allowing the digestion to continue for 10 to 15 minutes or longer. 
The ammonia formed in this process may be conveniently and quickly dis- 
tilled into 2 c.c. of 0.05 N hydrochloric acid contained in a second test-tube, 
which need not be as heavy as the Pyrex tube and should be graduated at 25 
c.c.^ Add to the hydrolyzed blood filtrate a dry pebble, 2 c.c. of saturated 
borax solution and a drop or two of parafl&n oil; firmly insert the rubber stop- 
per carrying both delivery tube and receiver (care being of course taken that 
the delivery tube dips below the acid solution in the receiver), and boil 
moderately fast over a microburner for 4 minutes. The size of the flame 
should never be cut down during the distillation, nor should the boiling be so 
brisk that the emission of steam from the receiving tube begins before the 
end of 3 minutes. At the end of 4 minutes slip off the receiver from the rub- 
ber stopper and lower the receiver in such a way that the delivery tube rests 
along the side of the receiver about midway between the 25 c.c. graduation 

^ One should never use for this decomposition the Pyrex tubes employed in the non- 
protein determinations, unless they are first washed with nitric acid to remove the mercury 
films, which are prone to collect on the walls of the tubes. After treatment with the nitric 
acid, wash thoroughly with water and follow by alcohol and thorough drying. Mercury 
prevents the action of urease and must be removed. 

- The composition of the phosphate solution used is as follows. To 140 grams of sodium 
pyrophosphate (U.S. P.) add 20 grams of glacial phosphoric acid and make up to i liter. 
Instead of this solution, one may use a molecular ortho-phosphate solution, consisting of 
a mixture of molecular monosodium phosphate and molecular disodium phosphate solutions 
in the proportion of l-i of the former to % of the latter. 

^ The urease solution is prepared as follows: Transfer to a 200 c.c. flask or bottle about 
3 grams of permutit powder. Wash this by decantation, once with 2 per cent, acetic acid, 
then twice with water. Add to the moist permutit in the flask 100 c.c. of 30 per cent, 
alcohol. Then introduce 5 grams of jack bean meal (which may be obtained from the 
Arlington Chemical Co., Yonkers, N. Y.) and shake for 10 minutes. Filter and collect 
the filtrate in three or four different small clean bottles. Set one aside for immediate use, 
as it will remain serviceable for at least i week at ordinary room temperature if not exposed 
to direct sunlight. Place the other bottles in the ice box, where they wiU remain active for 
3 to 5 weeks. The addition of the buffer solutions (phosphate mixtures) is essential when 
this solution is used, as the decomposition of urea is never dependable otherwise. This 
action is, according to Folin, twofold: the}^ not only accelerate the decomposition of the 
urea, but they prolong the acting period of the urease to a great extent. 

^ The distilling apparatus recommended by Folin and Wu consists of the original Pyrex 
test-tube, to which is attached a second test-tube, which serves as a receiver. This latter 
is held in place b}- means of a rubber stopper in the side of which has been cut a fairly deep 
notch to permit the escape of air (and some steam). The rubber stopper, serving as a hold- 
er for the receiver, fits quite loosely to the delivery tube by means of which the test-tubes 
are connected. In the beginning of the distillation, the delivery tube must dip below the 
acid in the receiver; near the end this delivery tube is raised about midway between the 
graduation mark on the test-tube and the top of the tube. 



476 DIAGNOSTIC METHODS 

mark and the top of the receiving tube. Continue the distillation for i 
minute more and rinse off the lower outside part of the delivery tube with a 
little water. Cool the distillate with running water, dilute to about 20 c.c. 
and add 2.5 c.c. of the Nessler solution discussed on page 472. Fill to the 
25 c.c. mark with water and compare in the colorimeter with a standard con- 
taining 0.3 mg. of N (in the form of ammonium sulphate) in a. 100 c.c. flask 
and Nesslerized with 10 c.c. of the Nessler solution. Both the standard 
and unknown should always be Nesslerized as nearly simultaneously as prac- 
ticable. Multiply 20 (the height of the standard in mm.) by 15 and divide 
by the colorimetric reading to get the urea nitrogen per 100 c.c. of blood. 
To convert the nitrogen values into terms of urea, multiply the N by 2.143. 

Should one prefer to use the process of aeration proceed as follows: De- 
compose the urea of the blood filtrate as described above. Add i or 2 c.c. 
of 10 per cent, sodium hydroxid solution and aspirate the ammonia (as 
described on page 240) into a test-tube graduated at 25 c.c. and containing 
2 c.c. of 0.05 N hydrochloric acid. Precaution must be taken that the 
rubber tubing connections are well washed with water before being used, as 
new tubing is coated with talcum powder which may contain ammonia. 
The method, after aeration, is as above described. 

Further, if one prefers, the autoclave method may be adopted. To 5 
.c.c. of blood filtrate in a 75 c.c. Pyrex test-tube is added i c.c. of normal 
acid; the mouth of the tube is covered with tin-foil, and the test-tube with 
contents is placed in an autoclave and heated at i5o°C. for 10 minutes. 
Allow the autoclave to cool below ioo°C. before opening. Now distill off 
the ammonia as given above, using 2 c.c. of 10 per cent, sodium carbonate 
•solution instead of the borax employed in the first process discussed. If 
desired, the ammonia may be driven off by aeration as usual. The pro- 
cedure from this point is the same as given above. 
Amount of Urea. 

There is much confusion in the hterature regarding the amount of urea in 
the blood, as the methods used have been numerous and the urea has been 
reported both in terms of urea and of urea nitrogen. The older methods 
must be regarded as inaccurate, so that figures obtained from them cannot 
be accepted at the present time. To obtain comparative figures for these 
■different factors, multiply the urea by 0.467 to get the urea nitrogen, or mul- 
tiply the urea nitrogen by 2.143 to derive the urea. 

Folin and Denis, in their analysis of the blood of sixteen normal individuals, 
report that the urea nitrogen varies only between 11 and 13 mg. (approxi- 
mately 24 to 28 mg. of urea) per 100 grams of blood. As these specimens of 
blood were collected in the morning 3 to 6 hours after breakfast, the results 
are to be considered as essentially those from a fasting person. During the 
digestion of a full meal containing meat, these values are probably increased 
by 2 to 3 mg. or more of urea nitrogen. Other workers, using the same 
method, report higher figures for the normal urea nitrogen, the values running 
in some cases as high as 20 to 25 mg. in 100 c.c. of blood. These higher fig- 
ures are perhaps above the true normal values, but they represent the usual 



THE BLOOD 477 

findings in the everyday run of clinical material. The urea nitrogen nor- 
mally constitutes about 50 per cent, of the total non-protein nitrogen. In 
some cases it may be a trifle higher, but almost never is it found to be lower. 
During digestion the percentage relationship is, of course, increased. 

In a general way it is to be expected that variations in the amount of urea 
nitrogen will parallel those of the total non-protein nitrogen. As urea is ex- 
creted largely through the kidney, serious disturbance of the renal paren- 
chyma should be associated with retention of urea. As a matter of fact, it 
has been found that in severe cases of nephritis, accompanied with symptoms 
of uremia, retention of urea is marked, reaching in some cases as high as 250 
mg. urea nitrogen per 100 c.c. of blood and constituting 80 per cent, or more 
of total non-protein nitrogen. It is evident, therefore, that marked retention 
of urea is a positive indication of almost complete renal insufficiency. A large 
part of the kidney parenchyma may be pathological and yet no retention of 
urea occur, providing the remaining renal tissue is sufficient to take care of 
the ordinary systemic requirements. In other words, cases either of inter- 
stitial or diffuse nephritis may show little or no retention of urea. Should 
urea retention become marked in such cases, uremia is impending owing 
to the excessive work put upon the remaining functionating renal cells. 
Chronic passive congestion of the kidney almost never shows urea retention. 
Further, one kidney may be so extensively diseased as to be incapable of 
functionating and yet no retention of urea occur, as the vicarious activity 
of the remaining kidney prevents any accumulation. For this reason, this 
determination of blood urea can have no importance in the study of uni- 
lateral renal diseases. 

A number of French investigators have advocated a comparison of the 
concentration of urea as excreted in the urine with its concentration in the 
blood, beheving that a very close relationship exists. Ambard, as a result of 
his studies, has formulated two general laws with reference to this point as 
follows: (i) When the concentration of urea in the urine is constant, the 
quantity of urea excreted in the urine varies proportionately to the square of 
the concentration of urea in the blood. (2) When the concentration of urea 
in the blood remains constant, the quantity excreted in the urine varies in- 
versely as the square root of the concentration in the urine. From these 
laws, corrected for the weight of the individual (70 kilos being the standard) 
and the average concentration of urea in the urine (25 grams per liter), 
Ambard formulated his " uremic constant'' or " coefficient,'' which is as follows: 

Ur 

X (constant) = I Z^ rp 

in which Ur = grams of urea per liter of blood; D = grams of urea in 24-hour 
specimen of urine; F = weight of patient in kilos; C = concentration or 
grams of urea per hter of urine. 

Normally this coefficient of Ambard is 0.06 to 0.09, being practically un- 
affected by variations in diet as is the amount of urea in the blood. Where 



478 DIAGNOSTIC METHODS 

the amount of urine excreted is very low, this constant tends toward higher 
values. In cases of severe nephritis a large increase in this coefficient is ob- 
served associated with a relative increase in the concentration of urea in the 
blood and a decrease in the elimination. It would seem, therefore, that this 
coefficient of Ambard might be of considerable value in the study of cases 
showing httle actual retention of urea, as far as the absolute amount of the 
urea is concerned, but in which actual evidence of nephritis is present. In 
other words, may not a renal insufficiency be indicated by this coefficient, 
when the amount of urea nitrogen is practically normal? Although we have 
been somewhat loath to accept the conclusions of Ambard and his coworkers, 
recent studies, especially by McLean and Selhng, would indicate that an in- 
creased coefficient may be found in nephritic cases, which do not show any 
appreciable retention of urea. In the severe types of nephritis, which are 
tending toward uremia, this coefficient will be markedly increased, just as are 
the total non-protein nitrogen and urea. 

As McLean points out, the determination of the amount of urea nitrogen, 
or of the total nonprotein nitrogen in the blood, is not entirely dependable 
as a measure of excretory activity. The normal concentration of urea in 
the blood is not fixed, but varies within wide Hmits in response to variations 
in nitrogen and fluid intake. As a matter of fact, the larger proportion of 
individuals with disturbed urea function have concentrations of urea in their 
blood which are below the upper limits in normal individuals, so that the line 
of demarcation between normal and abnormal subjects is not sharp. Blood 
urea figures alone, therefore, may call attention only to the more serious 
instances of disturbed function. In his work McLean has extended the laws 
of Ambard and introduced a relatively simple method for making clinical 
estimations of the state of the function which has to do with excretion of 
urea. ''It is important to state that investigations concerning urea excre- 
tion are especially valuable in the study of renal disease. For although the 
various functions of excretion by the kidney appear to be quite specific, so that 
no one test can be regarded as a measure of total renal function, nevertheless, 
the information obtained from a study of the urea function appears to be more 
general than that of any other." 

The ''Index of Urea Excretion,'' as originated by McLean^ is based on an 
ideal normal Ambard's coefficient of 0.08. Deficiency in urea function re- 
sults in higher values. These higher values are due to a change in the ratio 
which exists between the concentration of urea in the blood and the rate of 
its excretion. Such findings indicate deviation from the normal, but do not 
measure the extent of the functional change which is responsible for such 
deviation. In observations in human subjects it is not possible to keep 
conditions constant, but it is possible, by an appUcation of Ambard's laws, to 
measure the influence of their variations on the rate of excretion of urea. 
The ideal normal rate of excretion, under the conditions found in McLean's 
studies, at any time, is given a value of 100. The index then expresses, in 
direct percentage, the rate of excretion found, in terms of the rate of excre- 

1 Jour. Exper. Med., 1915, XXII, 212 and 366; Jour. A. M. A., 1916, LXVI, 415. 



THE BLOOD 479 

tion that a normal individual would develop under the same conditions as to 
concentration in the blood, concentration in the urine, and body weight. 
Ambard's laws depend on the constant relationship of four variables: (i) 
the concentration of urea in the blood; (2) the concentration of urea in the 
urine; (3) the rate of urinary excretion, which together with (2) gives the 
rate of urea excretion; and (4) the weight of the individual. When varia- 
bles which give an Ambard's coefficient of 0.08 (the figure chosen as the 
ideal normal) are substituted in the Ambard formula, the constant must have 
a value of 8.96 to give an index of 100. The new formula of McLean would 
then be 

Index of Urea Excretion = — Wri ^^rr <> — =100 (normal value). 

WtXUr^ 

In this formula D = Grams urea' excreted per 24 hours; C = Grams urea 
per liter urine; Ur= Grams urea per liter blood; Wt. = Body weight of 
individual in kilograms. f 

The rate of excretion is not actually determined for 24 hours, but for a 
shorter period, usually 72 minutes (3^^o of 24 hours), the calculation being 
made on a basis of grams per 24 hours. It is important to remember that it 
is not the actual twenty four hour excretion of urea that is essential, but 
the rate of excretion at the time of observation. McLean's procedure in 
the determination of this index is as follows: The patient is given from 150 
to 200 c.c. of water in order to insure a free flow of urine. One-half hour later 
he voids urine, in order to start with the bladder empty. The patient may be 
catheterized if necessary. The time of voiding is recorded to within i min- 
ute. . About 36 minutes later, from 7 to 10 c.c. of blood are withdrawn in 
the usual way and a few mg. of potassium oxalate added to prevent clotting. 
At the end of 72 minutes from the first voiding, the bladder is again emptied 
and the urine accurately measured. The patient takes no food or water 
during this period. If it is desired to carry out a simultaneous phenolsul- 
phonephthalein test, one may adopt the i or 2 hour period, instead of the 
72 minute one, half of the measured urine being used for the phenolsulpho- 
nephthalein reading, and the other half for the necessary analyses in connec- 
tion with this method, which are determination of blood urea, urine urea, and 
urine ammonia by methods previously outlined. Knowing these factors and 
the weight of the naked individual within i kilogram, the variables are 
inserted in the formula and the index calculated. This has been simplified 
by McLean, by the introduction of a special modified 10 inch slide-rule (ob- 
tainable from Keuffel and Esser Co., 127 Fulton St., New York), which 
permits a direct reading of the index. 

An index below 80 is to be considered as abnormal, though not necessarily 
seriously so. In renal disease, an index below 50 is indicative of a consider- 
able degree of impairment of functional ability. The lower the index, the 
greater the impairment to the kidney. However, a low index may be only 
temporary, as in passive congestion of heart failure or in acute nephritis. 
When observations are made under the proper conditions, values for the in- 
dex of less than 80 should never be found in normal individuals. Values 



480 DIAGNOSTIC METHODS 

above 100 are the rule and values up to 180 and 200 may occur. Much higher 
values may be obtained under certain pathologic conditions, associated with 
an increase in the rate of urea excretion. The figure obtained for the blood 
urea will give some indication as to the probable index, as low blood urea is 
associated with a high index and vice versa. As McLean points out, the 
application of the index is seriously interfered with when water excretion 
is greatly diminished, so that he make it a general rule not to attempt to 
apply this index when a rate of urinary outflow equal to at least 500 c.c. in 
24 hours cannot be attained. 

Addis -and Watanabe conclude, from their work, that factors other than 
the concentration of the blood urea must commonly intervene in the process 
of urea excretion. They call attention to our lack of definite knowledge of 
the factors governing the excretion of urea by the kidneys. Lewis, also, 
finds that the normal variations are rather wide and are subject to many 
disturbing influences. McLean, in more recent work, concludes that urea 
retention, in the sense of a relatively increased concentration in the blood, 
is the result of increased resistance to the excretion of urea through the 
kidneys. The numerical value of Ambard's coefficient changes in urea re- 
tention, but the relation of the variable factors to one another remains other- 
wise unchanged. In certain individuals, with otherwise normal findings in 
regard to urea excretion, an unusual degree of constancy, to which McLean 
applies the term "fixation," has been found in the numerical results obtained 
by the application of Ambard's laws. These individuals are regarded as 
probably abnormal, but the pathological significance of the fixation has not 
been determined. 

As the liver is concerned to a considerable extent with the formation of 
urea, we might expect to find in cases of marked hepatic insufficiency a re- 
duction in the amount of urea in the blood, just as we do in the urine. How- 
ever, it is to be remembered that the liver is not wholly responsible for the 
conversion of ammonium salts and amino acids into urea, as Folin, Van 
Slyke and others have shown, so that it must be regarded as possible that a 
practically normal urea finding may obtain in the case of the blood and the 
urinary output be relatively much diminished. Further, even in extensive 
liver disease, such as cirrhosis and carcinoma, sufficient hepatic parenchyma 
may remain to continue the normal function of the liver. Recent work by 
Farr and Krumbhaar would seem to indicate that the urea nitrogen of the 
blood is actually not diminished in hepatic disease in contradistinction to the 
findings reported by various French workers.^ 

^ With reference to various points raised in this section on urea see Ambard, Compt. 
rend. Soc. de Biol., 1910, LXIX, 411 and 506; Assoc, franc, d'urol. Proc.-Verb., 1912, XV, 
518; Bull, et mem. Soc. med. des hop. de Paris, 1913, XXXVI, 765; Widal and Javal, Ibid., 
1911, XXXII, 627; Achard, Le role de I'uree en pathologie, Paris, 1912; Ambard and Weill, 
Jour, de Physiol, et de Path, gen., 1912, XIV, 753; Folin and Denis, Jour. Biol. Chem., 1912, 
XI, 87; Ibid., 1913, XIV, 29; Ibid., 1913, XV, 493; Rowntree and Fitz, Arch. Int. Med., 
1913, XI, 258; Farr and Austin, Jour. Exper. Med., 1913, XVIII, 228; Courmont, Boulud, 
Savy and Blanc-Perducet, Bull, et mem. Soc. med. d. hop. de Paris, 1913, XXXV, 259; 
Morel and Mouriquand, Ibid., 266; Chauffard, Ibid., 273; Brodin, Compt. rend. Soc. de 
Biol., 1913, LXXIV, 26; Legueu, Ambard and Chabanier, Arch. urol. Glin, de Necker, 
1913, I, 275; Legueu, Jour. d'UroL, 1914, V, I; Kholzoff, Russky Vrach, 1914, XIII, 465; 



THE BLOOD ' 48 1 

Uric Acid. 
Like many substances formed outside the kidneys but excreted by them 
in fairly large quantities, uric acid occurs in the blood in traces too small to 
yield distinct reactions and quantitative results with the usual tests as ap- 
plied to urine. The introduction of newer methods enables us to study much 
more closely the question of the normal uric acid values as well as the patho- 
logical variations. 

Method of Benedict. 

This method^ is a modification of that of Folin and Macallum and, also, 
of the later one of Folin and Denis. This consists in substituting a rapid and 
convenient procedure in place of the rather troublesome decomposition of 
the uric acid compound, with hydrogen sulphid by dissolving the precipitated 
uric acid with potassium cyanide solution. 

Technic. 

Ten to 20 c.c. of blood are withdrawn from the median basilic vein by 
means of an accurately graduated syringe, the barrel of which contains a 
little powdered potassium oxalate to prevent clotting. This blood is then 
added to 5 volumes (50 to 100 c.c.) of boiling o.oi N acetic acid in a casserole 
and the mixture heated to boiling for a moment. The casserole is then re- 
moved from the flame and 100 or 200 c.c. of boiling water (depending on the 
original volume of blood taken) are added. The mixture is then poured 
upon a folded filter, and the residue washed with 50 c.c. of boiling water 
(heated in the same casserole in which the original coagulation took place). 

Studzinski, Ibid., 678; Widal, Weill and Radot, Presse med., 1914, XXII, 409 and 565; 
Jour. d'UroI., 1914, V, 681; Tschertkoff, Dutsch, med. Wchnschr., 1914, XL, 1713; Marshall 
and Davis, Jour. Biol. Chem., 1914, XVIII, 53; McLean and Selling, Ibid., 1914, XIX, 31; 
Folin, Denis and Seymour, Arch. Int. Med., 1914, XIII, 224; Agnew, Ibid., 485; Tileston 
and Comfort, Ibid., 1914, XIV, 620; Farr and Krumbhaar, Jour. Am. Med. Assn., 1914, 
LXIII, 2214; Underbill, New York Med. Jour., 1915, CII, 662; Klinkert, Nederl. Tijdschr. 
V. Geneesk., 1915, II, 1567 and 1658; Foster, Tr. Assoc. Am. Phys., 1915, XXX, 305; Grossi, 
Policlinico, 1916, XXIII, (sez. chir), 41; Kinney, N. Y. Med. Jour., 1916, CIV, 208; Payan 
and Mattel, C. R. soc. bioL, Paris, 1916, LXIX, 910; Scbwartz and McGill, Arcb. Int. Med., 

1916, XVII, 42; Hewlett, Gilbert, and Wickett, Ibid., XVIII, 637; Jones and Austin, Am. 
Jour. Med. Sc, 1916, CLII, 560; Addis and Watanabe, Jour. Biol. Chem., 1916, XXIV, 203; 
Ibid., XXVII, 249; Ibid., XXVIII, 251; Ibid., 1917, XXIX, 391 and 399; Arch. Int. Med., 

1917, XIX, 507; Lewis, Ibid., i; Watanabe, Am. Jour. Med. Sc, 1917, CLIV, 76; Nakagawa, 
Brit. Jour. Surg., 1917, IV, 386; Smith, Jour. A. M. A., 1917, LXVIII, 278; Pearce, Jour. 
Lab. and Clin. Med., 191 7, II, 590; Goto, Jour. Exper. Med., 191 7, XXV, 693; McLean, Ibid., 
XXVI, 181; Stengel, Austin, and Jonas, Arch. Int. Med., 1918, XXI, 313; Frisselland Vogel, 
Ibid., XXII, 56; Frothingham, Ibid., 74; Kast and Wardell, Ibid., 581; Kast and Killian, 
Proc. Soc. Exp. Biol, and Med., 1919, XVI, 141; Myers and Fine, Jour. Biol. Chem., 1919, 
XXXVII, 239; Reimann and Hartman, Am. Jour. Physiol., 1919, L, 82; McLean and De 
Wesselow, Quart. Jour. Med., 1919, XII, 347; Albert, Biochem. Ztschr., 1919, XCIII, 
89; Merklen and Kudelski, Bull. et. mem. soc. hop. de Paris, 1919, XXI, 604; Ameuille, 
Presse Med., 1919, XXVII, 189; Rouzaud, C. R. soc. biol. Paris, 1919, LXXXII, 727; 
Bevier and Shevky, Am. Jour. Physiol., 1919, L, 191. 

With reference to methods for determination of urea in blood see Folin and Pettibone, 
Jour. Biol. Chem., 1912, XI, 507; Folin and Denis, Ibid., 527; Marshall, Ibid., 1913, XIV, 
283; Ibid., 1913, XV, 287 and 495; Van Slyke and Cullen, Ibid., 1914, XIX, 211; Jour. Am. 
Med. Assn., 1914, LXII, 1558; Olivieri, Riv. osped., 1914, IV, 221; Kristeller, Ztschr. f. 
exper. Path. u. Therap., 1914, XVI, 496; Siebeck, Deutsch. Arch. f. klin. Med. ,1914, 
CXVI, 58; Neumann, Biochem. Ztschr., 1915, LXIX, 134; Combe and Levi, Rev. med. de la 
Suisse Rom., 1915, XXXV, 413. 

^ Jour. Biol. Chem., 1915, XX, 629. ^ 
31 



482 DIAGNOSTIC METHODS 

The total filtrate is now transferred to a casserole and boiled rapidly down to 
a volume of about 25 c.c. This solution is poured into a small flask roughly 
marked to indicate a volume of 50 c.c. The contents of the casserole are 
washed quantitatively into the flask with the help of two or three portions 
of water, heating the water to vigorous boiling, and rubbing the sides of the 
casserole with a rubber-tipped stirring rod each time. The total volume in 
the flask should not exceed 50 c.c. after addition of the washings. The tur- 
bid solution in the flask is now cooled thoroughly under running water and 2 
c.c. of colloidal iron solution (Merck's "Dialyzed Iron," 5 per cent, solution) 
are added while the flask is gently rotated. The mixture is then filtered 
through a small folded filter into a 100 c.c. Jena Florence flask, the residue 
on the filter being twice washed with distilled water. The filtrate obtained 
here should be clear and colorless. The solution is now boiled down to a 
volume of I to 2 c.c, then carefully poured into a small centrifuge tube, and 
the flask washed out with three portions of water (i to 2 c.c. each), heating 
each to boiling in the flask and shaking thoroughly prior to transferring it 
to the centrifuge tube. The contents of the tube, which should have a 
volume of 5 to 10 c.c, are now cooled and treated with 20 drops of ammoni- 
acal silver magnesium solution. ^ The contents of the tube are mixed thor- 
oughly with the help of a narrow stirring rod, and the tube is then centrif uged 
for I or 2 minutes. The supernatant fluid is then poured off as completely 
as possible, and to the residue in the tube are added one or two drops of 5 
per cent, potassium or sodium cyanide solution and the mixture is thoroughly 
stirred for a moment. A few drops of water are then added and the mixture 
is again stirred. One or two c.c. of the uric acid reagent of Folin and Macal- 
lum^ are then added (i c.c. if the bulk of the original precipitate was very 
small, otherwise 2 c.c) and, correspondingly, 5 or 10 c.c of 20 per cent, 
sodium carbonate solution are then added, and the colored solution is washed 
quantitatively into a 25 or 50 c.c flask and diluted to the mark with distilled 
water. Compare the color of this solution, in a colorimeter, with that of one 
of the standards given below and calculate the results as there explained. 
In determining this point of dilution of the unknown solution, one compares 
roughly the color obtained at this point with that of the standard solution 
treated with the uric acid reagent and sodium carbonate solution. This 
standard solution should be prepared just prior to the addition of the car- 
bonate to the unknown solution. Should the color of the unknown solution, 
when diluted to 25 c.c be too deep for that of the standard solution, dilute 
to 50 cc in another volumetric flask, which should be at hand ready for this 

^ This solution has the following composition: 70 c.c. of 3 per cent, silver lactate solution; 
30 c.c. of magnesia mixture; and 100 c.c. of concentrated aqueous ammonia. The solu- 
tion should be filtered before use. The magnesia mixture used in this solution is made as 
follows: Dissolve 17.5 grams of crystallized magnesium sulphate and 35 grams of ammonium 
chlorid in about 100 c.c. of water; add 60 c.c. of concentrated aqueous ammonia, and dilute 
o 200 c.c. 

2 The uric acid reagent is made as follows: To 750 c.c. of water add 100 grams of sodium 
tungstate and 80 c.c. of syrupy (86%) phosphoric acid. Boil gently for 2 hours, using a 
reflux condenser to prevent undue concentration. Cool the solution and dilute it to i 
liter in a volumetric flask. Two c.c. of this solution gives the maximum color obtainable 
with I mg. of uric acid. 



THE BLOOD 483 

emergency. The same statement applies with reference to the dilution to 
100 c.c. if necessary. 

As Folin remarks, "No other detail in the colorimetric determination of 
uric acid has presented such difhculties as has the problem of finding a suitable 
standard." The first standard solution used, and one which gives accurate 
results but which is not at all permanent, is a solution of pure uric acid in 
lithium carbonate solution, prepared as follows: Two hundred and fifty mg. 
of Kahlbaum's pure uric acid are washed into a 250 c.c. volumetric flask by 
means of 25 to 50 c.c. of water. Add 25 c.c. of a 0.4 per cent, lithium carbon- 
ate solution, shaking at intervals for an hour before diluting. Make up to 
250 c.c. in the flask and allow to stand for a few minutes before using. Each 
c.c. of this solution contains i mg. uric acid. In using this solution for com- 
parison with the unknown solution, of uric acid, take i c.c. of the standard 
solution, add 5 to 10 c.c. of water, 2 c.c. of the uric acid reagent and 20 c.c. of 
saturated sodium carbonate solution. Make up to the mark in a 100 c.c. 
volumetric flask. This solution is not advisable for the general laboratory 
as it necessitates the preparation of a fresh solution almost each time a uric 
acid determination is to be made and, hence, causes a needless loss of ma- 
terial owing to the fact that the solution does not keep longer than a few days. 

Folin and Denis have, more recently, advised a permanent solution of uric 
acid, which meets the requirements in a general way but which must itself be 
standardized against the above solution of uric acid in lithium carbonate to ob- 
tain its colorimetric value. This solution is made by combining uric acid with 
formaldehyd as follows: One gram of uric acid is placed in a liter volumetric 
flask and dissolved by the addition of 200 c.c. of a 0.4 per cent, solution of 
lithium carbonate. To the solution are added 40 c.c. of 40 per cent, formalde- 
hyd solution and the mixture is shaken and allowed to stand for a few minutes. 
Acidify the clear solution with 20 c.c. of normal acetic acid and dilute the 
mixture to the mark with water. The solution should renlain perfectly clear 
and the next day (but not before) should be standardized against a freshly 
prepared lithium carbonate solution of uric acid as given above. The color 
produced by 5 c.c. of this solution corresponds very nearly to that obtained 
from I mg. of uric acid. The colorimetrici reading obtained for this solution 
when compared against i mg. of pure uric acid, treated with the uric acid re- 
agent and the sodium carbonate solution as above, is used as the standard 
value corresponding to i mg. of pure uric acid. It is seen, therefore, that this 
solution does not possess the reactivity corresponding to the actual amount of 
uric acid in it, so that its value must be determined against known uric acid 
solutions. This standardized permanent solution gives accurate figures and 
may be used as a routine. 

Benedict and Hitchcock^ have found a standard solution for use in this 

test which seems to be an accurate and extremely permanent one. It has 

been used by the writer many times and is especially recommended for all com- 

^ Jour. Biol. Chem., 1915, XX, 619. Curtman and Freed (Ibid., 1916, XXVIII, 89) 
advise the use of 25 c.c. of 4% boric acid solution, instead of the glacial acetic acid of 
Benedict and Hitchcock's standard, for use in the cold months. In warm weather this 
boric acid solution is inferior to the acetic acid solution. 



484 DIAGNOSTIC METHODS 

parisons in this test. The solution is as follows: Nine grams of pure crys- 
tallized disodium hydrogen phosphate, together with i gram of crystallized 
sodium dihydrogen phosphate are dissolved in 200 to 300 c.c. of hot water 
and the solution is filtered, if it was not perfectly clear. Make up the filtrate 
to a total of about 500 c.c. with hot water and pour this clear solution upon 
exactly 200 mg. of pure uric acid suspended in a few c.c. of water in a liter 
volumetric flask. Agitate the mixture for a few moments until the uric 
acid is completely dissolved and cool. Now add from a buret exactly 1.4 
c.c. of glacial acetic acid and make up the contents of the flask to the liter 
mark with water. Mix the contents thoroughly and add about 5 c.c. of 
chloroforrii to prevent development of bacteria or moulds. Five c.c. of this 
solution contain exactly i mg. of uric acid. If there is any question of the 
standardizing of this solution, it may be checked up against standard solu- 
tions of uric acid in lithium carbonate once every month or so. 

The calculation of the results obtained in the colorimetric determinations 
is not difl&cult. Treat the unknown solution and the standard solution with 
the prescribed amounts of uric acid reagent and sodium carbonate solution, 
diluting the standard (i mg. of uric acid) to 100 c.c. and the unknown to 
25, 50 or to 100 c.c. as stated above. Set the standard solution at 20 mm. 
and compare the colors. In making the calculation use the following formula: 

n^ = milligrams of uric acid per 100 c.c. of blood. 

In this formula 20 represents the standard reading, R gives the reading in mm. 
of the unknown solution, V represents the volume to which the unknown is 
diluted, and W represents the volume of blood taken for the determination. 

Method of Folin and Wu. 

This method^ is included in the system of blood analysis advocated by 
these authors and may be followed in routine work, even though no other 
blood constituents are to be determined. In this method the uric acid is pre- 
cipitated directly from the filtrate from the blood proteins. 

Reagents Necessary. 

1. A new standard uric acid solution. This is prepared as follows: 
Make up i to 3 Hters of a 20 per cent, solution of sodium sulphite and let it 
stand over night. Filter the solution. Dissolve i gram of pure uric acid 
in 125 c.c. to 150 c.c. of 0.4 per cent, lithium carbonate solution and dilute to 
a volume of 500 c.c. Transfer 50 c.c. of this solution, corresponding to 100 
mg. of uric acid to each of a series of volumetric liter flasks. Add 200 to 
300 c.c. of water, then 500 c.c. of filtered 20 per cent, sodium sulphite solution, 
and finally make up to volume and mix well. Fill a series of 200 c.c. bottles 
and stopper very tightly with rubber stoppers. The solution in a bottle 
which is opened daily will keep for at least 3 to 4 months. 

2. A 10 per cent, solution of sodium sulphite. This is prepared from the 
excess of the 20 per cent, solution used in preparing the standard uric acid 
solutions. It may be kept in small tightly stoppered bottles. 

1 Jour. Biol. Chem., 1919, XXXVIII, 100. 



THE BLOOD 485 

3. A 5 per cent, solution of sodium cyanide, to be added from a buret 
when used in the test. 

4. A 10 per cent, solution of sodium chlorid in o.iN hydrochloric acid. 

5. The uric acid reagent of Fohn and Macallum, mentioned under the 
previous test. 

6. A solution of 5 per cent, silver lactate in 5 per cent, lactic acid. 

Technic. 

To 10 c.c. of blood filtrate from the tungstic acid precipitation described 
under Non-protein Nitrogen in each of two centrifuge tubes add 2 c.c. of a 
5 per cent, solution of silver lactate in 5 per cent, lactic acid, and stir with a 
fine glass rod. Centrifuge; add a drop of the silver lactate solution to the 
supernatant solution, which should be almost perfectly clear and should 
not become turbid when the last drop of silver solution is added. Remove 
the supernatant liquid by decantation as completely as possible. Add 
to each tube i c.c. of a solution of 10 per cent, sodium chlorid in o.i N hydro- 
chloric acid and stir thoroughly with a glass rod. Then add 5 to 6 c.c. of 
water, stir again, and centrifuge once more. By this treatment the uric acid 
is set free from the precipitate. Transfer the two supernatant liquids by 
decantation to a 25 c.c. volumetric flask. Add i c.c. of a 10 per cent, sodium 
sulphite solution, 0.5 c.c. of a 5 per cent, solution of sodium cyanid, and 3 
c.c. of a 20 per cent, solution of sodium carbonate. 

Prepare simultaneously two standard uric acid solutions as follows: 
Transfer to one 50 c.c. volumetric flask i c.c. and to another 50 c.c. flask 2: 
c.c. of the standard uric acid sulphite solution mentioned above. To the 
first flask add also i c.c. of 10 per cent, sodium sulphite solution. Then add 
to each flask 4 c.c. of the acidified sodium chlorid solution, i c.c. of the so- 
dium cyanid solution, and 6 c.c. of the sodium carbonate solution. Dilute 
with water to about 45 c.c. When the two standard solutions and the 
unknown have been prepared, add 0.5 c.c. of the uric acid reagent to the 
unknown and i c.c. to each of the standards, and mix. Let stand for 10 
minutes, fill to the mark with water, mix, and make the color comparisons. 

In connection with the calculation of the amount of uric acid in the un- 
known, it is to be noted that the blood filtrate taken (20 c.c) corresponds to 
2 c.c. of blood, that the standard is diluted to twice the volume of the un- 
known, and that the standard used contains o.i or 0.2 mg. of uric acid. The 
blood filtrate from blood containing 2.5 mg. of uric acid will be just equal in 
color to the weaker standard. Twenty times 2.5 divided by the reading of 
the unknown gives, therefore, the uric acid content of the blood when the 
weaker standard is set at 20 mm. Obviously, if the stronger standard is 
necessary and is set at 20 mm., the calculation is changed by the substitution 
of 5 for the 2.5 of the weaker standard. If a still weaker standard is needed,, 
add 5 c.c. of 20 per cent, sodium carbonate solution to 25 c.c. of the regular 
weaker standard and dilute to 50 c.c. This latter corresponds to the color 
obtained from 1.25 mg. of uric acid per 100 c.c. of blood. ^ 

^ For other methods see Maase and Zondek, Miinch. med. Wchnschr., 1915, LXII,. 
1 105; Host, Ztschr. f. physiol. Chem., 1916, XCV, 88; Bogert, Jour. Biol. Chem., 1917,, 
XXXI, 165; Curtman and Lehrman, Ibid., 1918, XXXVI, 157. 



486 DIAGNOSTIC METHODS 

Amount of Uric Acid. 

There is no question but that uric acid is present in appreciable amounts in 
normal blood, although such recognized investigators as Brugsch and Schitten- 
helm state that such is not the case. This divergence of opinion is due to the 
fact that the older methods were far from adequate. In one of their reports, 
Folin and Denis give the normal uric acid values of human blood as varying 
from I to 2.5 mg. per 100 grams of blood. Concerning these figures they state 
that "we are not prepared to say that they represent the full variations." 
A rise in the uric acid values undoubtedly occurs following a meal rich in nu- 
clein-containing food, but the extent of this " digestive rise " has not been defi- 
nitely established. Weintraud has reported the presence of 5 mg. of uric 
acid per 100 c.c. of blood during digestion. This must point to the fact that 
the kidneys have a limited power of excreting uric acid when a surplus is 
suddenly poured into the blood from the digestive tract. Sooner or later, 
however, this digestive accumulation is adjusted and the blood findings be- 
come more nearly the figures given above as normal. Denis^ has shown that 
in normal men no increase in the circulating uric acid is produced by the 
ingestion of large quantities of purines. In persons suffering from renal 
insufficiency a more or less marked increase in the uric acid content of the 
blood is produced by high purine feeding. He concludes, therefore, that 
when the determination of uric acid in the blood is undertaken as a diag- 
nostic test, the insistence on a preliminary period during which no purine- 
containing foods are consumed is unnecessary except where a renal 
insufficiency exists. It would thus seem that the normal kidney reacts 
toward an excess of uric acid in a way essentially similar to that in which 
it conducts itself toward an excess of urea and is able to excrete the excess 
of uric acid, thereby keeping the circulating uric acid at the same level as 
that obtained when only endogenous uric acid is to be excreted. While Folin 
and Denis have reported increased amounts of uric acid in pathological con- 
ditions, especially in nephritis, it is interesting to note "that there is 
apparently no relationship between the amount of uric acid and the amount of 
urea or total non-protein nitrogen in human blood." While the urea and total 
non-protein nitrogen of the blood are inversely proportional to the general 
efficiency of the kidney, the significance of the uric acid is less clear. Accord- 
ing to Folin, we may have four distinct classes of blood with reference to uric 
acid: (i) blood in which both uric acid and non-protein nitrogen are present 
in normal amounts; (2) blood in which with normal amount of uric acid we 
have greatly increased amount of non-protein nitrogen; (3) blood giving 
abnormally high uric acid values with normal amounts of non-protein nitro- 
gen; and (4) blood in which abnormally large amounts of both uric acid and 
non-protein nitrogen are present. 

The condition of increase of uric acid in the blood is known as uricacidemia 

or lithemia. In cases of severe nephritis, in which the eliminatory power of 

the kidney is greatly reduced, a relatively marked increase in the uric acid of 

the blood is observed. This increase in nephritis reaches the highest values 

^ Jour. Biol. Chem., 1915, XXIII, 147. 



THE BLOOD 487 

for the uric acid of the blood, being as high as 10 mg. in some cases, and is 
3,ssociatedwith high non-protein nitrogen and urea values. As Myers and 
Fine^ state, the kidney normally concentrates the creatinin 100 times, the 
urea 80 times, but the uric acid only 20 times. It would seem, therefore, 
that high uric acid values would be shown much earlier than those of 
creatinin and urea which fact should be of value in early diagnosis. Chace 
and Myers^ report uric acid values as high as 22.4 mg. per 100 c.c. of blood 
in cases of fatal chronic interstitial nephritis. An increase in the uric acid 
is, also, observed in all conditions associated with increased nuclein decom- 
position. In this group we find leukemia, pneumonia, carcinoma and 
severe febrile conditions. Further, an increase is noted in practically all 
cases of chronic lead poisoning, due, perhaps, to the associated renal 
degeneration. 

For some time following the teaching of Garrod, an accumulation of uric 
acid in the blood was held to be pathognomonic of gout. That this idea is 
erroneous is seen from the list of conditions in which an increase is observed. 
In fact these other diseases frequently show a uric acid value much higher 
than is ever reached in gout. The usual increase in gout runs from 3 to 7 mg. 
According to Magnus-Levy, there is no increase of uric acid in the blood pre- 
ceding or during the gouty attack, but a marked accumulation follows the 
seizure. Folin's findings would seem to indicate that in gout there is almost 
invariably an abnormally high uric acid value while the total non-protein 
nitrogen is within normal limits. In non-gouty arthritis the blood is not 
infrequently high in uric acid, but most cases have abnormally high non- 
protein nitrogen. Hence, for differential diagnosis in a case of suspected 
gout, the uric acid value is of no importance in itself, but must be taken in 
connection with a high or low non-protein nitrogen or urea.^ 

Ammonia. 

Normally the blood contains ammonium compounds to a very slight ex- 
tent, the limits not having yet been determined. This ammonia arises partly 
from the normal pancreatic digestion and finds its way into the circulation as 
a precursor of urea. Moreover, it is especially absorbed into the blood as a 
product of bacterial putrefactive processes in the large intestine, as Folin and 

ijour. Biol. Chem., 1919, XXXVII, 239. 

2 Jour. A. M. A., 1916, LXVII, 929; see, also, Myers, Fine, and Lough, Arch. Int. Med., 
1916, XVII, 570. 

^ See Folin and Macallum, Jour. Biol. Chem., 191 2, XIII, 363; Folin and Denis, Ibid., 
469; Ibid., 1913, XIV, 29 and 95; Brugsch and Kristeller, Deutsch. med. Wchnschr., 1914, 
XL, 746; Weiss, New York Med. Jour., 1914, C, 180; Adler and Ragle, Boston Med. and 
Surg. Jour., 1914, CLXXI, 769; Krocher, Deutsch. Arch. f. klin. Med., 1914, CXV, 380; 
Steinitz, Deutsch. med. Wchnschr., 1914, XL, 953; Fine and Chace, Jour. Pharmacol, and 
Exper. Therap., 1914, VI, 219; Liefmann, Ztschr. f. Kinderhkde., 1915, XII, 227; Benedict 
and Hitchcock, Jour. Biol. Chem., 1915, XX, 619; Benedict, Ibid., 629 and 633; Folin and 
Denis, Arch. Int. Med., 19 15, XVI, 33; Masse and Zondek, Miinch. med. Wchnschr., 1915, 
LXII, 1 105; Host, Norsk Mag. f. Laegevidensk, 1915, LXXVI, 11 12; Denis (Jour. Phar- 
macol, and Exper. Therap., 191 5, VII, 255) shows the increased elimination of uric acid 
following use of salicylates; Jorgensen, Ugeskr. f. Laeger, i9i5,LXXVII, 1429; Fine, Jour. 
A. M. A., 1916, LXVI, 2051; Pratt, Am. Jour. Med. Sc, 1916, CLI, 92; N. Y. State 
Jour. Med., 1916 XVI, 531; Fine. Ibid., 541; Watanabe, Am. Jour. Med. Sc, 1917, CLIV, 
76; Kingsbury and Sedgwick, Jour. Biol. Chem., 1917, XXXI, 261; Slemons and Bogert, 
Ibid., XXXII, 63; Baumann, Hausmann, Davis, and Stevens, Arch. Int. Med., 1919, 
XXIV, 70; Upham and Higley, Ibid., 557. 



488 DIAGNOSTIC METHODS 

Denis have shown. As a further source one finds the metabohc activities so 
adjusted that any excess of acids, introduced into the system from without or 
produced within the system by increased protein decomposition, is neutral- 
ized up to a certain point by a corresponding increase in the ammonia pro- 
duced. It is, in thrs case, to be regarded as a stage in the deamidization of 
the proteins, being formed when certain nitrogenous groups are Hberated 
from the amino acids, which are hnked together to form the protein molecule. 
We are justified, therefore, in assuming that a preceding increase of ammo- 
nium compounds in the blood must obtain, to account for an excretion of 
such an excess of ammonia as we find in the urine under certain conditions 
already discussed. However, this point has not been actually demonstrated 
by experimentation. It is probably true that the actual amount of ammo- 
nium compounds in the systemic blood is almost infinitesimal under normal 
conditions, although the portal blood is rich in such elements. It is evident, 
therefore, that what ammonia does find its way into the blood is, to a con- 
siderable extent, rapidly removed therefrom, probably through the action 
of the ammonia-destroying function of the liver. Whether an hepatic in- 
sufficiency may be demonstrated by later researches on the ammonia of the 
blood is a matter of speculation. Rohde, in her excellent studies with the 
vividiffusion apparatus of Abel, has shown that there is no liberation of 
ammonia comparable to that which takes place under aseptic conditions in 
shed blood and, further, that no ammonia is formed from the diffusible con- 
stituents of the blood. 

The amount of ammonia in the circulating blood has, as mentioned above, 
not been definitely determined. The figures given by different workers are 
somewhat at variance, due, no doubt, to the fact that decomposition of ni- 
trogenous compounds begins as soon as the blood is drawn and, in conse- 
quence, the amount of ammonia may increase by leaps and bounds in a few 
hours. Hence, it is absolutely essential, in the use of any method for the 
determination of ammonia in the blood that the work be done rapidly. In 
his earlier studies of the ammonia of the blood, Folin showed that the am- 
monia nitrogen of the blood from the mesenteric vein of the large intestine of 
cats ranged from 0.24 to 1.6 mg. per ico c.c. of blood; while that from the 
portal vein of the same animals showed a range of a trace only to 0.22 mg. per 
100 c.c; and that from the small intestine ranges from 0.05 to 0.77 mg. per 
100 c.c. It is, probably, true that the upper limit of the ammonia nitrogen 
■ of human blood is i mg. per 100 c.c. Thus, Gettler and Baker, using the 
method of Folin and Denis, find an average of between 0.4 and 0.75 mg.; 
Barnett, with a somewhat modified technic, finds an average in five normal 
men of between 0.07 and o.i mg. Folin and Denis find less than i mg. and 
Folin states that when precautions are taken to render the potassium oxalate 
and permutit, used in the method of Morgulis and Jahr, ammonia-free 
" the amount of ammonia obtained from protein-free blood filtrates is prac- 
tically nothing and only a greenish yellow color is obtained on Nesslerizing." 
Morgulis and Jahr find the normal values, by their method to range from 0.14 
to 0.30 mg. per 100 c.c. of blood. 



THE BLOOD 489 

While the normal ammonia is relatively insignificant in amount, yet 
quite an appreciable amount is shown by several workers in pathologic con- 
ditions. While, as stated above, ammonia is rapidly formed when blood 
stands after being drawn, yet the increase noted in certain cases can hardly 
be assumed to be entirely due to this factor. McNeil and Levy state that 
"In no instances, however, have we found the ammonia content of sterile 
normal blood to rise above 3 mg. per 100 c.c. even after prolonged standing, 
nor have any of the observers mentioned above found the rise in ammonia 
to exceed this Hmit after standing." Hence they consider that under any 
circumstances a finding of over 3 mg. per 100 c.c. may be safely taken as 
abnormal. In a recent contribution by Gad-Andersen, it is shown that the 
ammonia nitrogen of muscle tissue varies from a value of 0.8 mg. per 100 c.c. 
of blood (the determination being made at once) to 5.0 mg. per 100 c.c. 2 
hours later. He beheves that the concentration of ammonia is the same in 
muscle and blood and that the increase in ammonia, shown on allowing the 
material to stand before determining the ammonia, is due to the decomposition 
of the urea of the muscle. This would seem to refute the statements of Sum- 
ner as well as those of Marshall and Davis, although these latter workers 
conclude that the concentration of urea in blood and tissues is approximately 
the same even though they did not find the relatively large amounts of 
ammonia reported by Gad-Andersen. McNeil and Levy have reported an 
extensive study of the blood ammonia in several series of cases with the 
following results: In chronic diseases of the liver, a range of ammonia from 
45 to 60 mg. per 100 c.c; in myocarditis, 30 to 130 mg. per 100 c.c; in acid- 
osis from various causes, 40 to 330 mg. per 100 c.c; in nephritis, 40 to 100 
mg. per 100 c.c of blood. 

Owing to the small amount of ammonia present in the blood and its rapid 
formation from other substances on standing, the methods advanced for its 
determination are not entirely satisfactory. The recent technic of Mor- 
gulis and Jahr would appear to be very satisfactory, were it not that cer- 
tain amino acid derivatives present in the blood filtrate are absorbed by the 
permutit used and introduce a variable factor in the Nesslerization process. 
For this reason we advise the method of Folin and Denis, which is much older 
and gives good comparative figures, although it has been shown by Henriques 
and Christiansen that the amount of ammonia recovered may vary with the 
rapidity of the aeration and the temperature. The method of Barnett is 
cumbersome and requires the most assiduous efforts to prevent variations in 
the titer of the solutions used in the micro-titration of the ammonia. As 
such determinations do not, at present, offer any clinical advantages, the 
writer is content with reference to the hterature.^ 

1 Folin, Ztschr. f. physiol. Chem., 1902, XXXVII, 161; Medwedew, Ibid., i9ii,LXXII, 
410; Folin and Denis, Jour. Biol. Chem., 1912, XI, 161 and 527; Taylor and Ringer, Ibid., 
1913, XIV, 407; Jacobson, Ibid., 1914, XVIII, 133; Fiske and Karsner, Ibid., 381; Rohde, 
Ibid., 1915, XXI, 325; Henriques and Christiansen, Biochem. Ztschr., 1916, LXXVIII, 165; 
Barnett, Jour. Biol. Chem., 191 7, XXIX, 459; Barnett and Addis, Ibid., XXX, 41; McNeil 
and Levy, Jour. Lab. and Clin, med., 1917, II, 509; Ibid., Ill, 18; Morgulis and Jahr, 
Jour. Biol. Chem., 1919, XXXVIII, 4355 Folin, Ibid., XXXIX, 259; Gad-Andersen, 
Ibid., 267. 



490 ' DIAGNOSTIC METHODS 

Creatinin. 

The number of creatinin and creatin determinations in blood recorded in 
the hterature is very small. Only within the last few years has the impor- 
tance of a study of the variations in the creatinin content of the blood been 
recognized. FoHn and Denis report that they have found no specific creatinin 
retention, the figures in some 200 cases indicating that the human kidneys 
remove creatinin from the blood with remarkable ease. The normal crea- 
tinin content of the blood appears to lie between i and 2 mg. per 100 c.c. of 
blood, while the creatin value runs as high as 5 or 8 mg. It is, no doubt, 
true that a retention of creatinin occurs, practically speaking, only in neph- 
ritis. In such cases we find the creatinin values paralleHng those of non- 
protein nitrogen and urea, high retention being observed only in the severe 
cases which show a tendency toward uremia. Myers and Fine report as high 
creatinin values as 33 mg. per 100 c.c. of blood in cases of this type. Myers 
and Lough beheve the estimation of blood creatinin to be of considerable 
prognostic value, figures from 2.5 to 3 mg. being suspicious, those from 3 to 
5 mg. being regarded as decidedly unfavorable, while values over 5 mg. prob- 
ably indicate an early fatal termination. Veeder and Johnston show, from 
a study of 75 cases of normal children as well as those with scarlet fever 
and other clinical conditions, that the normal values of creatinin range 
from 0.58 to 3.44 mg., while the range in febrile conditions is from 1.08 to 
3.82 mg.; the normal creatin is rarely over 5 mg. with no specific relation 
obtaining between creatinin and creatin. The values reported by Gettler 
and Baker for creatinin are remarkably low, being o.i to 0.5 mg.; while 
creatin shows a range of t,.o to 6.5 mg. Since the introduction of the FoHn 
picric acid method for the determination of creatinin, it has been shown 
by several workers that the results by this method were somewhat higher 
than the true values. Folin and Doisy, Wilson and Plass, and Hunter and 
Campbell have shown that picric acid gives high values, due to the fact that 
the acid is impure so that erroneous colorations arise when the solutions 
are treated with alkalies. Hunter and Campbell, working on the distribu- 
tion of creatinin and creatin between the plasma and corpuscles find the 
following: Creatinin of normal human blood plasma ranges between 0.7 
and 1.3 mg. per 100 c.c, it being practically certain that this is distributed 
through the corpuscles and plasma in uniform concentration; creatin of the 
blood is chiefly concentrated in the corpuscles, the range being 6 to 9 mg. per 
100 c.c, while the plasma shows not more than 0.4 to 0.6 mg. per 100 c.c; the 
values of creatin in the whole blood average about 3 mg. per 100 c.c. Green- 
wald and McGuire give as the normal values, 3 to 5 mg. per 100 c.c of total 
creatinin, with a normal creatin value of 1.4 to 3.7 mg. per 100 cc They 
regard anything over 5 mg. of total creatinin as suspicious, 6 as pathological, 
and 10 very serious. Denis, using his proposed new method, finds the total 
creatinin of human blood ranging from 4 to 8 mg. per 100 c.c. in a series of 
pathological cases. It is evident, therefore, that the earlier figures, as re- 
ported for creatinin and creatin, must be regarded as high due to the errors 
in the methods used. By the use of the new method of Folin and Wu (out- 



THE BLOOD 49 I 

lined below) the total creatinin value for normal blood is given as 6 mg. per 
100 c.c, which is quite at variance to that of the earlier method of Folin.^ 
From the distribution of the non-protein nitrogenous compounds in the blood 
and urine, it would appear that of the three constituents usually estimated 
creatinin is normally the most readily, and uric acid the least readily, elim- 
inated, with urea standing in an intermediate position. It seems reasonable 
to conclude that when a noticeable retention of creatinin has occurred, the 
functional condition of the kidney has been markedly impaired. 

Method of Folin and Wu. 
This method^ was introduced in order to avoid the errors incident to the 
determination of creatin when picric acid was used as a protein precipitant. 
While, perhaps, no more accurate than the methods of Greenwald and Mc- 
Guire or of Denis, yet it is more convenient than these as it forms part of the 
system of blood analysis, which permits the determination of practically all 
the non-protein nitrogenous constituents with one precipitation of protein. 

Determination of Preformed Creatinin. 
Transfer 25 (or 50) c.c. of a saturated solution of picric acid (purified by 
the method of Folin and Doisy, see urine page 256) to a small clean flask, 
add 5 (or 10) c.c. of 10 per cent, sodium hydrate solution and mix. Trans- 
fer 10 c.c. of the tungstic acid blood filtrate (discussed under non-protein 
nitrogen) to a small flask or to a test tube. Transfer 5 c.c. of the standard 
creatinin solution^ to another flask and dilute this to 20 c.c. Then add 5 c.c. 
of the alkaline picrate solution, freshly prepared as above, to the blood filtrate, 
and 10 c.c. to the diluted standard creatinin solution. Let stand for 8 to 
10 minutes and make the color comparison in the usual manner, never 
omitting first to ascertain that the two fields of the colorimeter are equal 
when both cups contain the standard creatinin picrate solution. This color 
comparison should be completed within 15 minutes from the time the alka- 
line picrate was added. 

1 Folin and Denis, Jour. Biol. Chem., 191 2, XII, 141; Ibid., 1914, XVII, 475, 487, and 
493; Neubauer, Miinch. Med. Wchnschr., 1914, LXI, 857; Myers and Fine, Jour. Biol. 
Chem., 1915, XX, 391; Foster, Arch. Int. Med., 1915, XV, 356; Myers and Lough, Ibid., 
XVI, 536;'Woods, Ibid., 577; Veeder and Johnston, Am. Jour. Dis. Child., 1916, XII, 136; 
Gettler and Baker, Jour. Biol. Chem., 1916, XXV, 214; McCrudden and Sargent, Ibid., 
XXVI, 527; Plass, Bull. Johns Hopk. Hosp., 191 7, XXVlII, 137; Folin and Doisy, Jour. 
Biol. Chem., 1917, XXVIII, 349; Gettler, Ibid., XXIX, 47; Wilson and Plass, Ibid., 413; 
Hunter and Campbell, Ibid., XXXII, 195; Ibid., 1918, XXXIII, 169; Greenwald and Mc- 
Guire, Ibid., XXXIV, 103; Denis, Ibid., XXV, 513; Myers and Killian, Proc. Soc. Exp. 
Biol, and Med., 1918, XVI, 41- . 

2 Jour. Biol. Chem., 1919, XXXVIII, 98. 

^ One standard creatinin solution, suitable both for creatinin and creatin determina- 
tions in blood, can be made as follows: Transfer to a liter flask 6 c.c. of the standard creatinin 
solution described on page 258 (which contains 6 mg. of creatinin); and 10 c.c. of normal 
hydrochloric acid, dilute to the mark with water, and mix. Transfer to a bottle and add 
four or 5 drops of toluene or xylene. 5 c.c. of this solution contains 0.03 mg. of creatinin, 
and this amount plus 15 c.c. of water represents the standard needed for the vast majority 
of human blood, for it covers the range of i to 2 mg. per 100 c.c. In the case of unusual 
bloods representing retention of creatinin, take 10 c.c. of the standard plus 10 c.c. of water- 
which covers the range of 2 to 4 mg. of creatinin per 100 c.c. of blood; or 15 c.c. of the stand- 
ard plus 5 c.c. of water, by which 4 to 6 mg. may be estimated. By taking the full 20 c.c 
volume from the standard solution at least 8 mg. may be estimated; but when working 
with such blood, it is probably advisable to substitute 5 c.c. of blood filtrate plus 5 c.c. of 
water for the usual 10 c.c. o-f blood filtrate taken for the test. 



492 DIAGNOSTIC METHODS 

When the amount of blood filtrate available for the creatinin determina- 
tion is too small to permit repetition, it is of course advantageous or neces- 
sary to start with more than one standard. If a high creatinin should be 
encountered unexpectedly without several standards ready, the determina- 
tion may be saved by diluting the uaknown with an appropriate amount 
of the alkahne picrate solution — using for such dilution a picrate solution 
first diluted with two volumes of water — so as to preserve equality between 
the standard and the unknown in relation to the concentration of picric 
acid and sodium hydrate. 

Calculation. — The reading of the standard in mm. (usually 20) multiphed 
by 1.5, 3, 4.5, or 6 (according to how much of the standard solution was 
taken), and divided by the reading of the unknown in mm., gives the amount 
of creatinin in mg. per 100 c.c. of blood. In connection with this calculation 
it is to be noted that the standard is made up to twice the volume of the un- 
known, so that each 5 c.c. of the standard creatinin solution, while containing 
0.03 mg., corresponds to 0.015 mg. in the blood filtrate. 

Determination of Creatin plus Creatinin. 
Transfer 5 c.c. of the tungstic acid blood filtrate to a test-tube graduated 
at 25 c.c. Add i c.c. of normal hydrochloric acid. Cover the mouth of the 
test-tube with tin-foil and heat in the autoclave to i3o°C. for 20 minutes, or 
to i55°C. for ID minutes. Cool. Add 5 c.c. of the alkaline picrate solution 
and let stand for 8 to 10 minutes, then dilute to 25 c.c. The standard solu- 
tion required is 20 c.c. of creatinin solution in a 50 c.c. volumetric flask. 
Add 2 c.c. of normal hydrochloric acid and 10 c.c. of the alkaline picrate 
solution, and, after 10 minutes standing, dilute to 50 c.c. The preparation 
of the standard must, of course, have been made first so that it is ready for 
use when the unknown is ready for the color comparison. The height of the 
standard, usually 20 mm., divided by the reading of the unknown and mul- 
tiplied by 6 gives the ''total creatinin" in mg. per 100 c.c. of blood. In the 
case of uremic bloods containing large amounts of creatinin, i, 2, or 3 c.c. of 
blood filtrate, plus water enough to make approximately 5 c.c. are substitutes 
for 5 c.c. of the undiluted blood filtrate. The normal value for "total 
creatinin" by this method is about 6 mg. per 100 c.c. of blood. 

Amino Acids. 

Much discussion has, for long years, centered around the occurrence of 
amino acids in the blood. It is well known that these nitrogenous bodies, 
both of the mono- and di-amino types, are formed in the intestinal digestion of 
protein material. As the methods of research were for the most part inade- 
quate for the detection of these bodies, it was assumed that the blood did not 
contain them but that they were either resynthesized in passing through the 
absorptive process or were at once converted by the liver into urea. Both 
of these ideas have been shown to be fallacious, thanks to the work of Folin, 
Van Slyke and Meyer, Abderhalden (who was formerly one of the staunchest 
opponents of the presence of these bodies in the blood), and more recently of 
Abel, Rowntree and Turner. Many of these amino acids have been isolated 



THE BLOOD 493 

from the blood and identified by very careful chemical examination, so that 
there is no longer any doubt of their occurrence in the blood stream. Whether 
or not variations in the amounts of these bodies, leading to a hyper-amino- 
acidemia or to a hypo-amino-acidemia will prove to be of clinical value re- 
mains to be seen. The methods" available for this study are, as yet, not 
suitable for general clinical work, so that the writer will refrain from discus- 
sing them. Ellis, CuUen and Van Slyke report the amino-acid nitrogen of 
the blood as varying within certain definite limits (4.5 to 8.5 mg. per 100 c.c.) 
in different individuals and in the same individual at different times. Schlutz 
and Pettibone, in their studies of children, show that the average is 4 mg. per 
100 c.c. of blood in normal cases, while in pathological conditions the range 
is from 1.22 to 6.97 mg., cases of nephritis showing only 1.24 to 4.44 mg. 
Bock, using a slightly modified Greenwald method, finds the average normal 
adult figures to be 7.13 mg. In pathological cases, the range is from 4.5 to 
30 mg. per 100 c.c, the most pronounced increase being noted in nephritis.^ 

(£) Carbohydrates. 
The presence of sugar in the blood, both of normal and of abnormal 
types, is a well demonstrated fact. Whether this sugar is in the free molecu- 
lar state and is dialyzable or whether it is combined and non-dialyzable is 
still a matter of discussion. The discovery of jecorin (a combination of 
glucose with lecithin) by Drechsel indicates that such combinations occur 
in the blood, but it has by no means been proven that such compounds are the 
usual blood sugar. The work of Michaelis and Rona, Tachau, Gradwohl and 
Blaivas, along with others would seem to indicate that the sugar is in the free 
state; Lepine and Boulud, Kleiner, and others believe that the sugar is in 
combination. It is an interesting finding of Gradwohl and Blaivas that the 
amount of sugar is practically the same in the whole blood, plasma, and 
cells. Lepine states that certain blood samples may contain both free and 
combined sugar, both of which may be shown to dialyze. Michaelis and 
Rona point out that if both free and combined sugar exist side by side in the 
blood they are probably in equilibrium with one another; when the blood is 
dialyzed the free sugar will pass out thus destroying the equilibrium and 
forcing the decomposition of a part of the combined sugar. While other sub- 
stances, such as uric acid and creatinin, are present in the blood and show 
a redujcing action, yet there can be no question of the presence of true sugar 
itself. The sugar usually present is glucose, but maltose is occasionally 
found in the blood of nursing mothers, while levulose and pentose may occur 



1 In this connection see Folin, Jour, Biol. Chem., 191 2, XI, 87; Van Slyke, Ibid., 191 1, 
IX, 185; Ibid., 191 2, XII, 275; Ibid., 1913, XVI, 121 and 125; Van Slyke and Meyer, Ibid., 
1912, XII, 399; Abderhalden, Ztschr. f. physiol. Chem., 1913, LXXXVIII, 478; Kaplan, 
New York Med. Jour., 1913, XCVII, 1172; Ibid., 1913, XCVIII, 157; Kaplan and McClel- 
land, Ibid., 1012; Abel, Rowntree and Turner, Jour. Pharmacol, and Exper. Therap., 1914, 
V, 611; Ellis, Cullen and Van Slyke, Jour. Am. Med. Assn., 1915, LXIV, 126; Gyorgy and 
Zunz, Jour. Biol. Chem., 191 5, XXI, 511; Schlutz and Pettibone, Am. Jour. Dis. Child., 
1915, X, 206; Pettibone and Schlutz, Jour. A. M. A., 1916, LXVII, 262; Ross, Jour. 
Biol. Chem., 1916, XXVII, 45; Bock, Ibid., 1917, XXVIII, 357; Ibid., XXIX, 191; 
Okada, Ibid., 1918, XXXIII, 325. 



494 DIAGNOSTIC METHODS 

in the blood and urine after intake of large amounts of food containing 
them.^ 

Glucose. 

The occurrence of glucose in the blood is termed glycemia. This hexose 
is found normally in quantities varying from 0.4 to 1.5 parts per 1000, the 
average being probably about 0.8 per 1000 or, in other words, 0.08 gm. per 
100 c.c. of blood. Under pathological conditions this amount may increase 
to as high as i.i gm. per 100 c.c, as reported by Myers and Bailey. Accord- 
ing to Claude Bernard, whenever the sugar of the blood reaches three parts 
per 1000, diuresis and glycosuria occur as evidences of the systemic effort to 
control the hyperglycemia. This figure is undoubtedly much too high and 
was obtained with methods which could hardly compare with those of the 
present day. The study of the ''renal threshold," by which is meant the 
height of the blood sugar level at which appreciable amounts of sugar are 
eliminated in the urine, by more exact methods shows that, when the blood 
sugar reaches 0.15 gm. per ido c.c, sugar will appear in the urine, although 
Hamman and Hirschman place this value between 0.17 and 0.18 gm. It is 
important to remember, in this connection, that this value is not to be re- 
garded as a constant, as many normal individuals may excrete sugar with a 
blood sugar value of 0.14 gm. or even less; while others may show even higher 
values than 0.18 gm. The usual test of the ''tolerance" for sugar, of ad- 
ministering 100 grams of glucose on an empty stomach and studying its 
effect both on the sugar of the blood and urine, indicates that only a moderate' 
hyperglycemia results and that this rapidly subsides, the blood sugar reaching 
normal values in about 2 hours. With a diabetic, however, we find that a 
lowered tolerance usually exists and that the hyperglycemia may reach more 
than 0.2 gm., the reaction lasting for 3 to 5 hours. Williams and Humphrey 
point out that there is no striking relation between the height of the renal 
threshold and the duration of the diabetes, although it would appear that the 
threshold tends to rise with the increasing duration of the disease. When the 
diabetes is mild or quiescent, the point at which the kidneys eliminate 
sugar is stationary; but when the disease becomes progressive, the threshold 
tends to rise, a rising threshold in the face of careful dietary treatment, 
being regarded as a serious prognostic sign. It will be seen, therefore, that 
a study of the "renal threshold" will, in a measure at least, enable us to differ- 
entiate between a true diabetes and a so-called "renal diabetes," although 
it is probably that no definite level can be fixed where a true diabetes begins 
owing to the variability of the normal value. Certain it is, however, that 
values about 0.17 should be regarded with great suspicion. 

While high values for blood sugar are observed in diabetes, we find, also, 
in nephritis that an increase, often above 0.2 gm. per 100 c.c obtains. Many ' 

^ Michaelis and Rona, Biochem. Ztschr., 1909, XVI, 60; Lepine and Boulud, Lyon 
Med., 1913, XLV, 997; von Hess and McGuigan, Jour. Pharmacol, and Exp. Therap., 
1914, VI, 45; Tachau, Ztschr. f. klin. Med., 1914, LXXIX, 421; Canavan and Dahlstrom, 
Wise. Med. Jour., 1916, XV, 151; Lepine, Jour, physiol. et path, gen., 1917, XVII, 377; 
Gradwohl and Blaivas, Jour. Lab. and Clin. Med., 191 7, II, 416; Kleiner, Jour. Biol. Chem., 
191 8, XXXIV, 471. 



THE BLOOD 495 

nephritics have a high renal threshold, while others, of apparently the same 
clinical type, have a normal threshold. The literature contains many studies 
of the blood sugar in nephritis and the general trend of the reports is toward 
an increase in the blood sugar. These values have, for the most part, been 
obtained by the methods of Lewis and Benedict, or the Myers and Bailey 
modification of this method, so that it is probable that the results are higher 
than those really obtaining, owing to the fact that creatinin, which is in- 
creased to an appreciable extent, reacts with the picric acid used in the test 
and tends to give false values for the blood sugar. ^ While Benedict does 
not beHeve the figures are influenced by creatinin, yet MorguHs and Jahr 
have shown, in their experiments of adding known amounts of creatinin 
to pure glucose solutions and employing the Benedict method, that quite 
an appreciable variation does result. By comparison with the new method 

^ Reicher and Stein, Ztschr. f. exper. Path. u. Therap., 191 2, X, 532; Herzfeld, Ztschr. 
f. physiol. Chem., 191 2, LXXVII, 420; Roily and Oppermann, Biochem. Ztschr., 1913, 
XL VIII, 50, 187, 200, 259, 268 and 471; Ibid., 1913, XLIX, 278; Jacobsen, Ibid., 1913, 
LVI, 471; Lampe and Strassner, Med. Klin., 1913, IX, 1462; Bing and Jakobsen, Ugesk. f. 
Laeger, 1913, LXXV, 1627; Borchardt and Benningson, Miinch. med. Wchnschr., 1913, 
LX, 2275; Autenrieth and Montigny, Ibid., 1914, LXI, 1671; Scott, Am. Jour. Physiol., 
1914, XXXIV, 271; Bergmark, Jahrb. f. Kinderhkde., 1914, LXXX, 373; Lichtwitz, 
Berl. klin. Wchnschr., 1914, LI, 1018; Broekmayer, Deutsch. med. Wchnschr., 1914, XL, 
1562; McLean, Jour. A. M. A., 1914, LXII, 917; Bing and Jakobsen, Deutsch. Arch. f. 
klin. Med., 1914, CXIII, 571; Menke, Ibid., 1914, CXIV, 209; Miiller, Ztschr. f. physiol. 
Chem., 1914, XCI, 287; Kahler, Wien. klin. Wchnschr., 1914, XXVII, 417; Dresel, Ztschr. 
f. exper. Path. u. Therap., 1914, XVI, 365; Epstein and Baehr., Jour. Biol. Chem., 1914, 
XVIII, 21; Wolf and Gutmann, Ztschr. f. klin. Med., 1914, LXXIX, 394; Hirsch, Biochem. 
Ztschr., 1915, LXX, 191; Ztschr. f. physiol. Chem., 1915, XCIII, 355; Ibid., 1915, XCIV, 
227; Ross and McGuigan, Jour. Biol. Chem., 1915, XXII, 407 and 417; Bass, Am. Jour. 
Dis. Child., 1915, IX, 6s; Wolff, Deutsch. med. Wchnschr., 1915, XLI, 6; Morita, Arch, 
f. exper. Path. u. Pharmakol., 1915, LXXVIII, 188; Lauritzen, Therap. d. Gegenw., 1915, 
LVI, 8 and 94; Hopkins, Am. Jour. Med. Sc, 1915, CXLIX, 254; Carlson and Ginsburg, 
Am. Jour. Physiol., 1915, XXXVI, 28c; Macleod and Pearce, Ibid., 1915, XXXVIII, 
415; Jahnson-Blohm, Upsala Lakaref . Forhandl., 1915, XX, 331; Strouse, Stein, and Wise- 
ley, Bull. Johns Hopk. Hosp., 1915, XXVI, 211; Geyelin, Arch. Int. Med., 1915, XVI, 
975; von Moraczewski, Biochem. Ztschr., 1915, LXXI, 268; Schumm, Ztschr. f. physiol. 
Chem., 1915, XCVI, 204; Pels, Jour. A. M. A., 1915, LXV, 2077; Niemann, Jahrb. f. 
Kinderhkde., 1916, LXXXIII, i; Graham, Jour. Physiol., 1916, L, 285; Lee and Scott, 
Am. Jour. Physiol., 1916, XL, 486; Lombroso, Atti accad. Lincei, 1916, I, 736; Schwartz, 
Heimann and Mahnken, Jour. Cutan. Dis., 1916, XXXIV, 159; Kleiner, Jour. Expert 
Med., 1916, XXIII, 507; McCrudden and Sargent, Arch. Int. Med., 1916, XVII, 465; 
Weston, Jour. Med. Res., 1916, XXXV, 199; Macleod, Jour. Lab. and Clin. Med., 1916, 
II, 112; Ryser, Deutsch. Arch. f. klin. Med., 1916, CXVIII, 408; Grote, Miinch. med. 
Wchnschr., 1916, LXIII, 1614; Rydgaard^ Ugesk. f. Laeger, 1916, LXXVIII, 2007; Nilsson, 
Upsala Lakaref . Forhandl., 1916, XXII, 107; Rogers, Boston Med. and Surg. Jour., 1916, 
• CLXXV, 152; Epstein and Aschner, Jour. Biol. Chem., 1916, XXV, 151; Underbill, Ibid., 
447, 463 and 471; Epstein, Reiss and Branower, Ibid., XXVI, 25; Murlin and Kramer, 
Ibid., XXVII, 481, 499 and 517; Hiller and Mosenthal, Ibid., XXVIII, 197; Kuriyama, 
Ibid., 191 7, XXIX, 127; Macleod and Hoover, Am. Jour. Physiol., 191 7, XLII, 460; 
Morriss, Bull. Johns Hopk. Hosp., 191 7, XXVIII, 140; Janney, Proc. Soc. Exper. Biol, 
and Med., 191 7, XV, 15; Hamman and Hirschman, Arch. Int. Med., 191 7, XX, 761; 
Epstein and Felsen, Am. Jour. Med. Sc, 1917, CLIII, 58; Cummings and Piness, Arch. 
Int. Med., 1917, XIX, 777; Denis, Aub. and Minot, Ibid., XX, 964; Mosenthal, 
Clausen, and Hiller, Ibid., 1918, XXI, 93; McCrudden and Sargent, Ibid., 252; Janney and 
Isaacson, Ibid., XXII, 160; Ishikawa, Mitt. a.d. Med. Fak. Tokyo, 1918, XIX, 497; Sak- 
aguchi, Hayashi, and Yezima, Ibid., XX, 61; Gettler and St. George, Jour. A. M. A., 1918, 
LXXI, 2033; Roth, Berl. klin. Wchnschr., 1918, LV, 589; McCrudden and Sargent, Jour. 
Biol. Chem., 1918, XXXIII, 387; Ross, Ibid., XXXIV, 335; Bandouin: Paris Med., 1919, 
IX, 346; Bailey, Arch. Int. Med., 1919, XXIII, 455; Williams and Humphreys, Ibid., 
537,. 546 and 559; Edwards, Ind. State. Med. Ass'n. Jour., 1919, XII, 296; Lindblom 
Hygiea, 1919, LXXXI, 753; Hamman and Hirschman, Bull. Johns Hopk. Hosp., 1919, 
XXX, 306; Chapin and Myers, Am. Jour. Dis. Child., 1919, XVIII, 555. 



496 DIAGNOSTIC METHODS 

of Folin and Wu, in which picric acid is not employed, it is seen that the 
figures either by the original Lewis and Benedict method, the modification 
of Myers and Bailey, or Benedict's last modification are higher. 
Method of Folin and Wu. 

In the original method of these authors, the blood sugar was determined 
colorimetrically by the help of the phenol reagent of Folin and Denis. In 
this determination, the errors due to creatin, creatinin, and uric acid are 
eliminated, while a new source of error is introduced, namely, that due to 
the so-called phenols. In the method to be discussed, this latter error has, 
also, been eliminated, so that we now possess a very exact method for this 
estimation. 

Solutions Necessary. 

1. Standard Sugar Solutions. — Three standard sugar solutions should 
be on hand: (i) a stock solution of i per cent, dextrose or invert sugar 
preserved with xylol or toluol; (2) a solution containing i mg. of sugar per 
10 c.c. (5 c.c. of the stock solution diluted to 500 c.c); (3) a solution contain- 
ing 2 mg. of sugar per 10 c.c. (5 c.c. of the stock solution diluted to 250 c.c). 

2. Alkaline Copper Solution. — Dissolve 40 grams of anhydrous sodium 
carbonate in about 400 c.c. of water and transfer to a hter flask. Add 7.5 
grams of tartaric acid and when the latter has dissolved, add 4.5 grams of 
crystallized copper sulphate. Mix and make up to a volume of i liter. If 
the chemicals used are not pure, a sediment of cuprous oxid may form in the 
course of i or 2 weeks. If this should happen, remove the clear supernatant 
reagent with a siphon or filter through a good quality filter paper. 

3. Molyhdate-Phosphate Solution. — Transfer to a liter beaker 35 grams 
of molybdic acid and 5 grams of sodium tungstate. Add 200 c.c. of 10 per 
cent, sodium hydrate and 200 c.c. of water. Boil vigorously for 20 to 40 
minutes so as to remove nearly the whole of the ammonia present in the 
molybdic acid. Cool, dilute to about 350 c.c. and add 125 c.c. of concen- 
trated (85 per cent.) phosphoric acid. Dilute to 500 c.c. 

Technic. 
Transfer 2 c.c. of the tungstic acid blood filtrate (corresponding to 0.2 
c.c. .of blood) discussed under non-protein nitrogen to a special blood sugar 
test-tube,^ and to two other similar test-tubes (graduated at 25 c.c.) add 
2 c.c. of standard sugar solution containing respectively 0.2 and 0.4 mg. 
of dextrose. To each tube add 2 c.c. of the alkahne copper solution. The 
surface of the mixtures must now have reached the constricted part of the 
tubes. If the bulb of the tube is too large for the volume (4 c.c.) a little, but 
not more than 0.5 c.c. of a diluted (i : i) alkaline copper solution may be 
added. If this does not suffice to bring the contents to the narrow part, 
the tube should be discarded. Test-tubes having so small a capacity that 

1 The special blood sugar test-tubes, as devised by Folin and Wu, may be obtained 
from Emil Greiner Company, 55 Fulton St., New York, or from the Arthur iH. Thomas 
Company, West Washington Square, Philadelphia. These tubes consist of a Pyrex tube 
graduated at 25 c.c. and constricted toward the bottom in such a manner as to form a bulb 
which will contain 4 c.c, the fluid rising just to the constricted portion of the tube when 
4 c.c. are present. The constriction is 8 mm. wide and 4 cm. in length. 



THE BLOOD 497 

4 c.c. fills them above the neck should also be discarded. Transfer the 
tubes to a boihng water bath and heat for 6 minutes. Then transfer them to 
a cold water bath and let them cool, without shaking, for 2 or 3 minutes. 
Add to each test-tube 2 c.c. of the molybdate-phosphate solution. The 
cuprous oxid dissolves rather slowly if the amount is large, but the whole, 
up to the amount given by 0,8 mg. of dextrose, dissolves usually within 2 
minutes. When the cuprous oxid is dissolved, dilute the resulting blue 
solutions to the 25 c.c. mark, insert a rubber stopper, and mix. It is essential 
that adequate attention be given to this mixing because the greater part of 
the blue color is formed in the bulb of the tube. The color comparisons are 
made in the usual manner. The depth of the standard in mm. multiplied by 
100 and divided by the reading of the unknown gives the sugar in mg. per 
100 c.c. of blood, when the lower standard is employed; while the depth of 
the standard must be multiplied by 200 when the solution containing 0.4 
mg. is employed. 

The two standards given, representing 0.2 and 0.4 mg. of glucose, are 
adequate for practically all cases, as they cover the range from about 70 to 
nearly 400 mg. of glucose per 100 c.c. of blood. In a series of 40 cases, re- 
ported by Fohn and Wu, the range of blood sugar was from 70 to 170 mg. per 
100 c.c. of blood. It is interesting to note that the parallel figures forcrea- 
tinin and sugar in the same cases ran as follows, notations being taken from 
different parts of the table: Total creatinin, 6.1 mg., sugar 119 mg.; total 
creatinin, 19.4 mg., sugar 99 mg.; total creatinin, 20.5 mg., sugar, 170 mg.; 
total creatinin, '27.2 mg., sugar, 157 mg. per 100 c.c. of blood. This newer 
method of Folin and Wu is to be regarded as superior to the earlier method 
as it eliminates the possibihty of reoxidation of the cuprous compounds as 
well as the error due to the so-called phenols in the blood filtrate.^ 

Glycogen. 

This polysaccharid undoubtedly appears in the blood singly or in com- 
bination with albuminous bodies. Salomon, Frerichs, Lepine, Ehrlich, and 
Gabritschewsky have reported it, while Caminer was unable to find it. Hup- 
pert obtained it in quantities ranging from 0.114 to 1.56 grams per 100 parts 
of blood. Much depends on the method used to isolate and determine this 
substance, as it is easily lost by careless manipulation. Certain properties of 
the granules found in the blood, both extra- and intracellularly, have led many 

1 Folin and Wu, Jour. Biol. Chem., 1920, XLI, 367. For the original method of Folin 
and Wu see Jour. Biol. Chem., 1919, XXXVIII, 106. For other methods and points in 
connection with these methods see Kowarski, Deutsch. med. Wchnschr., 1913, XXXIX, 
1635; Bang, Biochem. Ztschr., 1913, XLIX, 19; Miinch. med. Wchnschr., i9i3,LX, 2277; 
Dorner, Ztschr. f. klin. Med., 1914, LXXIX, 287; Kraus, Lancet, 1914, 1, 1249; Kaminura, 
Sei-I-Kwai, 1914, XXXIII, s^'} Gardner and Maclean, Biochem. Jour., 1914, VIII, 391; 
Maasa and Tachau, Ztschr. f. klin. Med., 1914, LXXI, i; Fitz, Arch. Int. Med., 1914, 
XIV, 133; Epstein, Jour. A. M. A., 1914, LXIII, 1667; Schaffer, Jour. Biol. Chem., 1914, 
XIX, 285; Lewis and Benedict, Ibid., 1915, XX, 61; Pearce, Ibid., XXII, 525; Kahn, 
Jour. A. M. A., 191S, LXIV, 241; Ryser, Deutsch. Arch. f. klin. Med., 1915, CXVIII, 316; 
Myers and Bailey, Jour. Biol. Chem., 1916, XXIV, 147; Morris, Jour. Lab. and Clin. Med., 
1916, I, 252; Macleod, Ibid., 445; McDanell, Ibid., 804; McGuigan, Ibid., 1917, II, 514; 
Ege, Ugesk. f. Laeger, 1917, LXXIX, 129; McGuigan and Ross, Jour. Biol. Chem., 1917, 
XXXI, 533; Benedict, Ibid., 1918, XXXIV, 203; Addis and Shevky, Ibid., XXXV, 43 
and 53; Egerer, Ibid., 565; Rohde and Sweeney, Ibid., XXXVI, 475; Benedict, Ibid., 
1919, XXXVII, 503; Morgulis and Jahr, Ibid., XXXIX, 119; Maclean, Biochem. Jour., 
1919, XIII, 135; de Wesselow, Ibid., 148; Yamakami, Am. Jour. Physiol., 1919, L, 177. 



498 DIAGNOSTIC METHODS 

to believe that glycogen is present as a characteristic in many conditions. 
There is much reason, however, to assume that these granules, which stain 
brownish with iodin, are not glycogen, but rather albuminous bodies of in- 
definite composition. For a discussion of this subject see the treatment of 
iodophiUa. 

. (F) Fats and Fatty Acids. 

The presence of free fats (palmitin, stearin, and olein) in the blood has 
been frequently observed both in health and disease. The relative quanti- 
ties of these fats vary in different animals and are subject to wide variation in 
the same animal under influence of diet. The total lipoid content of the 
blood under normal conditions would appear to be about 0.6 per cent., being 
higher under influence of digestion. 

The presence of an excess of free fat in the blood is referred to as lipemia. 
The physiologic variations are more notable than are the pathological, being 
observed after ingestion of a meal rich in fats, in breast-fed children, in preg- 
nant women, and in the obese. ^ Pathologically lipemia more or less perma- 
nent may be observed in various conditions, as in acute and chronic alcohol- 
ism, diabetes, arteriosclerosis, chronic nephritis, phthisis, carbon monoxide 
and phosphorous poisoning, gout, typhoid fever, fat embolism following 
injuries of the long bones, pneumonia, leukemia, acute infections, cachexia 
from inanition or malignant disease, hepatic diseases, and malaria. It has, 
therefore, little differential diagnostic value. 

The extent of the lipemia may vary from the presence of isolated fat 
droplets to the overloading of the blood to such a degree that it becomes 
salmon-colored, turbid, and milky. This fat may be either the normal fat 
which has been transported from different parts of the body or may be that 
abnormal to the body arising from excess of fat in the diet. It is possible that 
a marked decrease in the lipase of the blood may be a factor in the production 
of lipemia. The fat is soluble in ether and stains black with osmic acid and 
red with Sudan-Ill. Regarding the quantitative estimations of fat, I must 
refer to other sections, the Gephart-Csonka method^ having been applied 
to blood fats with good results. 

Concerning the presence of fatty acids in the blood little is known. Traces 
of volatile acids are sometimes present, but probably not as normal constitu- 
ents. Gaglio, Spiro, Irisawa, and Berlinerblau report sarcolactic acid as 
a normal finding, while Zweifel finds an excess of this acid in the blood and 
in the urine in cases of toxemia of pregnancy. An excess of fatty acids in the 
blood is known as lipacidemia. Von Jaksch found fatty acids in the blood 
in cases of diabetic coma, leukemia, acute yellow atrophy of the liver, while 
Hougounenq reports the presence of j8-oxybutyric acid in the cadaveric blood 

^ Hermann and Neumann, Biochem. Ztschr., 191 1, XLIII, 47; Bosco, Policlinico, 1914, 
XXI, 314 and 357; Sakai, Biochem. Ztschr., i9i4,LXII, 387; Klein and Dinkin, Ztschr. f. 
physiol. Chem., 1914, XCII, 302; Greenwald, Jour. Biol. Chem., 1913, XIV, 369; Ibid., 
1915, XXI, 29; Am. Jour. Med. Sc, 1914, CXLVII, 225; Bloor, Jour. Biol. Chem., 1914, 
Xyil, 377; Ibid., 1914, XIX, i; Ibid., 1915, XXII, 133; Imrie, Ibid., 1915, XX, 87. It 
is interesting to note that the lowest normal value for cholesterin obtained by Denis 
(0.167 per cent.) was found in a woman weighing 200 pounds. 

2 Jour. Biol. Chem., 1914, XIX, 521; see also, Csonka, Ibid., 1918, XXXIII, 401. 



THE BLOOD 499 

of a diabetic. It is rational to assume that in all those conditions associated 
with acidosis, fatty acids are present in the blood, as these may be detected in 
large amount in the urine in these states. Bloor and MacPherson^ show that 
the total fatty acids of whole blood averages 0.36 per cent., while in the 
plasma the value is 0.38 per cent. The figures of Csonka show a normal 
fatty acid average of 0.297 per cent., the unsaturated type forming 48 per 
cent, of the total. This latter worker shows, also, that the blood lipoid 
values in anemia are normal so long as the percentage of corpuscles remains 
above 3^^ the normal figures; below such percentage, the plasma shows a 
high total fat with low cholesterin and lecithin values. 

In recent times the variations in the amount of cholesterin in the blood 
have assumed considerable importance. It is, beyond doubt, true that 
cholesterin is present in increased amounts both associated with lipemia and 
not so associated. The cholesterinemia of the alimentary type appears to be 
dependent on the intake of cholesterin-containing food. While the figures 
for the amount of cholesterin in the blood vary to a sHght extent, a fairly 
accurate normal standard may be taken as 150 mg. per 100 c.c. of blood, 
although the average figures of Bloor are 0.21 per cent., while those of 
Denis range from 0.167 to 0.255 per cent, and of Gorham and Myers from 
0.13 to 0.19 per cent. 

A hyper-cholesterinemia obtains in pregnancy, just as does a lipemia. The 
cholesterin gradually rises during gestation, reaches its maximum in the 
last months of pregnancy and drops to normal in 8 to 10 days after delivery, 
irrespective of lactation. In the eclampsias of pregnancy very high values are 
noted. In chronic nephritis an increase is observed, the degree running 
parallel to the severity of the case. This depends on the averages taken as 
normal values, as Denis states that cholesterol remains at normal levels 
although as high values as 0.29 per cent, are reported by her while Gorham 
and Myers report a hypercholesterolemia, with values usually running about 
0.2 per cent. In arteriosclerosis an increase of greater or less extent is 
practically always observed. In diabetes meUitus and in tuberculosis one 
obtains relatively high values as a rule. Very high figures (up to 900 mg. 
per 100 c.c.) are observed in choleHthiasis, a point of some value in the study 
of pathogenesis of gall-stones. ^ 

1 Jour. Biol. Chem., 1917, XXXI, 79; Csonka, Ibid., 1918, XXXIII, 401. 

^Lifschiitz, Ztschr. f. physiol. Chem., 1907, L, 437; Ibid., 1907; LIII, 140; Ibid., 1908, 
LVIII, 175; Ibid., 1909, LXIII, 223; Biochem. Ztschr., 1913, LII, 206; Ztschr., f. physiol. 
Chem., 1914, XCI, 309; Ibid., 1914, XCII, 383; Ibid., 1914, XCIII, 209; Windaus, Ibid., 
1910, LXII, 174; Ibid., i9io,LXV, no; Hanes, Bull. Johns Hopkins Hosp., i9i2,|XXIII, 77; 
Weston, Jour. Med. Research, 1912, XXVI, 47; Weston and Kent, Ibid., 531; Henes, 
Deutsch. Arch. f. klin. Med., 1913, CXI, 122; Jour. Am. Med. Assn., 1914, LXIII, 146; 
Thaysen, Biochem., Ztschr., 1914, LXII, 89 and 115; von Czyhlarz and Fuchs, Ibid., 131; 
Antonelli, Policlinico, 1914, XXI, 341; Farini, Gazz. d. osp., 1914, XXXV, 993; Fischl, 
Wien. klin. Wchnschr., 1914, XXVII, 982; Anitschkow, Med. Klin., 1914, X, 465; Pribram, 
Zentralbl. f. inn. Med., 1914, XXXVI, 325;Quinan, Calif. State Jour. Med., 1914, XII, 
118; Weiss, New York Med. Jour., 1914, C, 180; Chauffardand Grigaut, Pressemed., 1914, 
XXI, 929; Milkowitsch, Russk. Vrach, 1914, XXII, 191 1; Cannata, Pediatria, 1915, 
XXIII, 161; HufiFmann, Zentralbl. f. Gynak., 191 5, XXXIX, S5; Henes, New York State 
1915, XV, 310; Mueller, Jour. Biol. Chem., 1915, XXII, i; Bloor, Ibid., XXIII, 317; 
Cruickshank and Tisdall, Jour. Mental Sci., 1916, LXII, 168; Macleod, Jour. Lab. and Clin. 
Med., 1916, I, 529; Luden, Ibid., 662; Small, Ibid., 809; Hymanson and Kahn, Am. Jour. 



500 DIAGNOSTIC METHODS 

An interesting fact is observed in the study of the blood of febrile cases, 
especially of typhoid fever, pneumonia, scarlet fever, etc. In such cases it 
is the rule to find a diminution of cholesterin, the higher the fever the less the 
cholesterin content of the blood. As the temperature diminishes, the chol- 
esterin gradually rises, returning to about the normal point as a rule. If the 
fever be long continued, the cholesterin rise in the period of defervescence 
goes quite appreciably above the normal values. 

Determination of Total Cholesterol. 
Method of Bloor.i 
Preparation of Sample. 

3 c.c. of whole blood, plasma, or serum are run slowly (a slow stream of 
drops) from a pipet into about 75 c.c. of a mixture of redistilled alcohol 
and ether (3 parts alcohol, i part etherj in a 100 c.c. graduated flask. The 
contents of the flask should be kept in motion during the process so that there 
is no clumping of the precipitated material. The contents of the flask are 
raised to boiling by immersion in a water bath (with constant stirring to avoid 
superheating), cooled to room temperature, filled to the mark with alcohol- 
ether, mixed, and filtered. The filtered liquid, if placed in a tightly stoppered 
bottle in the dark, will keep unchanged for a considerable time. By running 
the blood slowly into the large quantity of alcohol-ether, as directed above, 
the protein material is precipitated in finely divided form and, under these 
conditions, the short heating combined with the great excess of solvent is 
adequate for complete extraction of serum or plasma. With whole blood, 
the results are not quite so complete. 

Technic. 

10 c.c. of the above alcohol-ether extract are measured into a small 
flat-bottomed beaker and evaporated 7^/5/ to dryness on a water bath. Any 
heating, after drying is reached, produces a brownish color which passes into 
the chloroform used later and renders the subsequent determination difficult 
or impossible. This point must be remembered. The cholesterol is ex- 
tracted from the dry residue by boiling out three or four times with successive 

Obs., 1916, LXXIII, 1041; de Langen, Presse Med., 1916, XXIV, 332; Dewey, Trans. 
Chic. Path. Soc, 1916, X, 52; Henes, Surg. Wyn. and Obs., 1916, XXIII, 91; Goiialons, Sem. 
Med., 1916, XXIII, 408 and 639; Wahl and Richardson, Arch. Int. Med., 1916, XVII, 
238; Dewey, Ibid., 1916, XVII, 757; Dubin and Pearce, Ibid., XVIII, 426; Bloor, 
Jour. Biol. Chem., 1916, XXIV, 227; XXV, 577; XXVI, 417; Luden, Ibid., XXVII, 
273; Jour. Lab. and Clin. Med., 1917, III, 93 and 141; Denis, Jour. Biol. Chem., 1917, 
XXIX, 93; Bloor and MacPherson, Ibid., XXXI, 79; Bloor, Ibid., 575; Knudson, Ibid., 
XXXII, 337; Gorham and Myers, Arch. Int. Med., 1917, XX, 599; Csonka, Jour. Biol. 
Chem., 1918, XXXIII, 401; Luden, Jour. Lab. and Clin. Med., 1918, IV, 849; Giesens, 
Deutsch. Arch. f. klin. Med., 1918, CXXVII, 439; Stepp, Miinch. med. Wchnschr., 1918, 
LXV, 781; Burge and Reinhart, Ztschr. f. exp. Med., 1918, VII, 119; Bang, Biochem. 
Ztschr., 1918, XC, 383; Ibid., XCI, 104, iii, 122 and 224; Feigl, Ibid., XCII, 282; Bang, 
Ibid., 1919, XCIV, 359; Rothschild and Felsen, Arch. Int. Med., 1919, XXIV, 520; Luden, 
Jour. Lab. and Clin. Med., 1919, IV, 719. 

^ Jour. Biol. Chem., 1916, XXIV, 227. See, also, Csonka, Ibid., 431; Mueller, Ibid., 
XXV, 549; Hoover and Blankenhorn, Arch. Int. Med., 1916, XVIII, 289; Weston, Jour. 
Biol. Chem., 191 7, XXVIII, 383; Bloor, Ibid., XXTX, 437; Luden, Ibid., 463; Csonka, 
Ibid., 1918, XXXIV, 577; Bernhard, Ibid., XXXV, 15; Myers and Wardell, Ibid., XXXVI, 
147. 



THE BLOOD 50I 

small portions of chloroform and decanting into a 10 c.c. glass-stoppered, 
graduated cylinder, which has previously been cahbrated. (In this ex- 
traction an excess (4 or 5 c.c.) should be added each time and the mixture 
allowed to boil down to somewhat less than J^ its volume before decanting). 
The combined extracts, after cooling are then made up to 5 c.c. The solu- 
tion should be colorless, but not necessarily clear, since the slight turbidity 
clears up on adding the reagents. 

5 c.c. of a standard cholesterol solution in chloroform (containing 0.5 mg. 
of cholesterol) are measured into a similar 10 c.c. cyHnder. (It is conven- 
ient to make the cholesterol standard in two strengths: {a) the stock solution 
containing 0.2 gram of pure cholesterol in 200 c.c. chloroform; and {b) the 
standard solution for use, made by diluting 10 c.c. of the above to 100 c.c. 
with chloroform. 5 c.c. of this latter solution will contain 0.5 mg.) 

To each of the solutions in the 10 c.c. cylinders are added 2 c.c. of acetic 
anhydride and o.i c.c. of concentrated sulphuric acid (in order to produce 
the Liebermann-Burchard color reaction), the solutions are mixed by in- 
verting several times, then set away in the dark for 15 minutes, after which 
they are transferred to the cups of the colorimeter and compared as usual, 
setting the standard at 15 mm. The cement of the colorimeter cups is 
soluble in chloroform, so that a coating of plaster of Paris must be used 
to avoid solution. 

On account of the adverse criticism of this method by Weston and the 
determinations of Bloor, himself, on the use of standard solutions of choles- 
terin at different temperatures in making these color comparisons, Bloor 
has advised that this comparison be made at approximately 2 2°C., owing to 
the different behavior of the blood cholesterol as compared with the standard. 
It is recommended, therefore, that, instead of using the cholesterol solution 
as a standard, a solution of naphthol green B be employed as employed by 
Gorham and Myers. The standard solution of this dye is made by diluting 
2 c.c. of a 0.1 per cent, aqueous solution to 17 c.c. with distilled water, ob- 
taining a 0.0118 per cent, solution. This solution has a colorimetric strength, 
which is practically identical with that obtained from a 0.08 per cent, chloro- 
form solution of cholesterol and is, therefore, somewhat stronger than the 
standard solution of Bloor. The formula for computing the cholesterol, in 
the unknown solution, is given by Gorham and Myers as follows, R repre- 
senting the colorimetric reading of the unknown: 103 — R X 0.00847 = mg. 
of cholesterol in 5 c.c. of chloroform. 

This method of Bloor gives somewhat higher results than those by his 
first or saponification method. As Luden has shown, this may be due to 
certain bile derivatives other than cholesterin. She suggests that parallel 
determinations by the two methods be employed supplementing these by 
employing the dialyzation method of eliminating the bile salts and pigments 
as advocated by Hoover and Blankenhorn. The method of Myers and 
Wardell gives lower values than that of Bloor. 



502 DIAGNOSTIC METHODS 

Determination of the Cholesterol Esters. 
Method of Bloor and Kundson.^ 

20 c.c. of the alcohol-ether extract, obtained as in the method. above 
(or sufi&cient to contain about 0.5 mg. of combined cholesterol) are measured 
into a small flat-bottomed Erlenmeyer flask (50 c.c.) and i c.c. of a i per 
cent, alcoholic solution of digitonin is added. The whole solution is then 
evaporated just to dryness on the water bath. The digitonin combines with 
the free cholesterol forming digitonin cholesteride while the cholesterol 
present as ester is not affected. The dried residue in the flask is then ex- 
tracted by boiling out with successive small portions of petroleum ether 
(boiling point below 6o°C.), filtering the extract through a plug of fat-free 
cotton in the stem of a small funnel. In order to get a complete extraction 
with a small amount of solvent, 15 c.c. of the petroleum ether are first added, 
the flask is covered with a small watch glass (to prevent too rapid evapora- 
tion) and the whole boiled gently until about half the liquid is gone. The 
succeeding extractions are made in a similar manner with 7 to 8 c.c. of petrol- 
eum ether, which dissolves the cholesterol esters but not the digitonin pre- 
cipitate. The combined extracts are then evaporated just to dryness, and 
the esters taken up with chloroform as in the above method of Bloor. . These 
chloroform extracts, measuring slightly less than 5 c.c, are collected in a 10 
c.c. glass-stoppered cylinder, cooled, and made up to 5 c.c. The remainder 
of the determination is as given above for the determination of total choles- 
terol. 

(G) Acetone. 

The occurrence in the blood of demonstrable amounts of acetone is known 
as acetonemia. Deichmiiller and von Jaksch have found a substance giving 
the reaction of acetone in various conditions, especially in fevers, which find- 
ing has been confirmed by Reale. Whether acetone is a product of normal 
intermediary metabolism and, as such, is found in many physiologic and 
pathologic states, must be found in the section on Urine. ^ 
(H) Biliary Constituents. 

The conditions in which the biliary constituents, especially the pigments 
and acids, are found in the blood is termed cholemia.^ It is usually stated 
that these elements are not found in normal blood, but Croftan has shown 
that the bile acids are observed in the blood of healthy subjects. This is not 
unexpected as they are completely absorbed from the intestines and are re- 
excreted in the bile (Weintraud).* 

Pathologically, both the acids and the pigments are found in the blood 
in any condition associated with their appearance in the urine. Oftentimes 

1 Jour. Biol. Chem., 1916, XXVIII, 107; Ibid., 1917, XXIX, 7. 

2 See Marriott, Jour. Biol. Chem., 1914, XVIII, 507; Jour. Am. Med. Assn., i9i4,LXIII, 
397; Scheel, Ugesk. f. Laeger, 191 6, LXXVIII, 905; Moore, Am. Jour. Dis. Child., 1916, 
XII, 244; Van Slyke and Fitz, Jour. Biol. Chem., 191 7, XXXII, 495; Ibid., 1919, XXXIX, 
23- 

3 See Lehndorff, Prager med. Wchnschr., 1912, XXXVII, 495. 

* Hymans v. d. Bergh and Snapper (Deutsch. Arch. f. klin. Med., 1913, CX, 540) show 
that the serum always contains traces of bilirubin along with lutein. See, also, Whipple 
and Hooper, Jour. Exper. Med., 1913, XVII, 593 and 612. 



THE BLOOD 503 

they^may be found in the blood when no reaction for them is obtainable in the 
urine. While the bile acids exert marked toxic effects, such as hemolysis, 
the bihary pigments show, as Bouchard, de Bruin, Lugli, and Colosanti have 
demonstrated, certain harmful influences. Fh'nt ascribes the toxic effects 
observed in cholemia to cholesterin, but this idea needs confirmation. The 
most usual condition showing cholemia is jaundice. Whether the blood 
changes observed in the various types of jaundice are due, primarily, to the 
cholemia or to a primary hemolysis or to a combination of these effects as a 
result of intoxication is an unsettled question.^ 

(/) Inorganic Constituents. 

The inorganic composition of the blood shows quite a marked variation 
both under physiologic and pathologic influences. This variation applies 
both to the cellular and intracellular constituents of the blood. Changes in 
the molecular concentration as well as changes in the concentration of 
specific inorganic combinations are observed in the blood of the two sexes, as 
may be seen by consulting the table on page 447. 

Regarding the special significance of the different inorganic constituents 
little is known, but chemical analyses of the blood ash in health and disease 
have shown that the pathological variations are more important as regards 
the chlorids, phosphates, and the iron compounds. ^ 

Chlorids. 

Physiologically, a certain (about six parts per mille) concentration of 
sodium chlorid is necessary to hold the proteins in solution, as well as to main- 
tain the proper osmotic tension of the serum. The larger the proportion 
of plasma, the greater the percentage of chlorids in the blood. This is true 
only within certain limits as the NaCl-content remains practically constant, 
no matter how large an amount is ingested. This constant value is regulated 
by the increased or diminished renal excretion.^ 

In anemias the chlorids of the blood are usually high, yet, according to 
Limbeck, cases are occasionally found in which normal amounts of sodium 
chlorid obtain. In pneumonia, in which a diminished urinary excretion of 
chlorids is observed, the blood does not show an excess of chlorids but may 
even show a decrease owing to the effects of the exudative process. Dimin- 
ished ingestion of food, vomiting, diarrhea, and general exudative processes 
may be associated with a decrease in the chlorids of the blood, but this is 
merely temporary. In cases of marked nephritis, associated with retention 
of chlorids, the blood may show an excess of sodium chlorid. 

As discussed on page 477, certain French workers have formulated a 

^ See Pel, Deutsch. Arch. f. klin. Med., 1912, CVI, 239; also, Brugsch and Retzlaflf, 
Ztschr. f. exper. Path. u. Therap., 191 2, XI, 508; Barratt and Yorke, Ann. Trop. Med. and 
Parasitol., 1914, VIII, 509; Fischer, Ztschr. f. physiol. Chem., 1916, XCV, 78; Blankenhorn, 
Arch. Int. Med., 1917, XIX, 344; Bauer and Spiegel, Deutsch. Arch. f. klin. Med., 1919, 

exxix, 17- . 

2 See Weissbein and Aufrecht, Internat. Beitr. z. Path. u. Therap. d. Ernahrungsstor., 
1912, IV, 22. 

3 Bromberg (Jour. d'Urol., 1914, IV, 733) shows that the relation of the chlorids of the 
blood to those of the urine is normally i to 2 (hemo-renal index). Variations in this index 
are, he believes, the first indications of renal insufficiency. 



504 DIAGNOSTIC METHODS 

formula, as a working basis for a study of the excretion of urea in the urine. 
These same workers, especially Ambard and Weill, have applied this study 
to the excretion of sodium chlorid in human subjects. They have found 
that the same general laws, which were found applicable to urea, apply to 
sodium chlorid, with the exception that, while the excretion of urea occurs no 
matter how low its concentration in the blood plasma, there is a threshold for 
chlorid excretion, and, when the concentration in the plasma falls below this 
threshold value, excretion of chlorid practically ceases. In view of the 
fact that there is a wide difference in chlorid content of the corpuscles and 
plasma, plasma alone has been studied. The normal threshold value, as 
established by Ambard and Weill for'chlorid, is 5.62 grams per liter of plasma. 
Hence the sodium chlorid above 5.62 grams per liter determines the rate of 
excretion, the law being expressed as for urea as follows: 
Excess NaCl over 5.62 grams per liter of plasma 



jNaCl m 24 hours / ^^ ^, ^77 7 -. — ^ 

\ x^TT-^ — T^i V NaCl per liter of urme = Constant 

\ Wt. m kilos 

For practical use it appears best to calculate the plasma sodium chlorid from 
the rate of excretion, and to compare the calculated concentration with that 
actually found. The formula, as derived, with the use of values actually 
found for the constant in the above formula, reads 



Plasma NaCl = 5.62 + J Wt^^ 

^ ^ 79-33 

In which D equals grams of chlorid excreted in 24 hours; Wt. equals weight 
of the individual in kilograms; 70 equals the standard weight in kilos; C 
equals grams of sodium chlorid per liter of urine. If this formula be simpli- 
fied we obtain the following: 



Plasma NaCl = 5.62 + J-^^^ 
\4.2, 



Wt. 

Assuming that the laws for rate of excretion of sodium chlorid over the 
threshold remain constant in normal individuals, one may calculate the 
threshold by subtracting the calculated excess from the sodium chlorid 
actually found in the plasma, giving us the formula. 



Threshold = Plasma NaCl ' ^ ^^ 



4, 



4.23 X Wt. 

This formula is subject to error if the rate of excretion over the threshold 
varies. 

The method of collecting the specimens of blood and urine for the deter- 
mination of the sodium chlorid threshold, as well as for the urea index 
according to McLean have been given on page 479, to which the reader is 
referred. After removing the portion of the whole blood for urea determina- 
tion, the remainder is centrifuged at high speed to throw down the corpuscles 



THE BLOOD 505 

and the plasma is pipetted off. The total chlorids of both plasma and urine 
are then determined, and calculated as NaCl. McLean^ finds this threshold 
very constant at about 5.62 grams per liter, the actual average of his deter- 
minations being 5.61, the maximum variations being from 5.24 to 5.84 
grams. The normal and usual range of concentration of chlorids in the 
human plasms is from 5.62 to 6.25 grams of NaCl per liter or higher, accord- 
ing to the amount ingested. The rate of excretion of chlorids gives a basis 
for calculating the theoretical concentration of chlorids in the plasma. By 
comparing the concentration actually found with the theoretical concentra- 
tion, changes in the function of chlorid excretion may be studied. McLean^ 
reports that relatively increased concentration of chlorids in the plasma 
occurs especially in certain forms of cardiac and renal disease; under certain 
conditions, notably in fevers or in diabetes, or as the action of diuretics, 
the chlorid threshold may be temporarily or permanently lowered; failure 
to excrete chlorids in pneumonia is associated with a lowered concentration 
of chlorids in the plasma, the excretion beginning at the time this concentra- 
tion increases; edema is usually accompanied by a relatively increased con- 
centration of chlorids in the plasma. 

Determination of Plasma Chlorids. 
Method of Van Slyke and Donleavy. 

This method^ is a modification of the method of McLean and Van Slyke 
and has the advantage that the process may be simplified to a great extent 
and does away with the necessity of employing ''Blood Charcoal" as a 
protein precipitant. 

Solutions Necessary. 

1. Standard Silver Nitrate Solution. — This is an acid M/29.25 solution of 
silver nitrate containing picric acid as the protein precipitant, i c.c. of this 
solution is equivalent to 2 mg. of NaCl. The composition is as follows: 
5.812 grams of pure fused silver nitrate, 7.5 grams of purified picric acid, 
250 c.c. of nitric acid (sp. g. 1.42), dissolved in sufficient water to make i 
liter. As a check on the accuracy of this solution, it may be standardized 
against a known hydrochloric acid solution by the Volhard method or gravi- 
metrically. 

2. Standard Potassium lodid Solution.— This is a M/73.1 potassium iodid 
solution containing 2.27 grams of pure KI per liter, i c.c. of this solution 
is equivalent to 0.8 mg. NaCl, being so standardized that 2.5 c.c. is equivalent 
to I c.c. of the silver nitrate solution. This iodid solution is standardized 
against the silver nitrate solution as follows: 5 c.c. of the silver nitrate solu- 
tion are accurately measured out and mixed with 5 c.c. of the starch-citrate 
solution (see below) and the iodid solution is run in from a buret to a per- 
manent blue end-point. The amount required should be 12.65 c.c, 12.50 
c.c. being required to precipitate the standard silver solution and 0.15 c.c. 
additional to give the end-point. In this standardization, as also in the test- 

1 Jour. Exper. Med., 1915, XXII, 212. 

2 Ibid., 366. 

3 Jour. Biol. Chem., 1919, XXXVII, 551; McLean and Van Slyke, Jour. Am. Chem. 
Soc, 1915, XXXVII, 1128; Jour. Biol. Chem., 1915, XXI, 361. 



5o6 DIAGNOSTIC METHODS 

itself, only a permaaent and unmistakable blue is taken as the end-point. 
If the iodid is run in rapidly toward the end of the titration, iodid may be 
formed more rapidly than the silver nitrate precipitates it, and a false end 
point reached. It is wise to make these titrations against a background 
of yellow paper. The amount of excess KI required to produce the end- 
point varies directly as the volume of the solution; consequently it is desir- 
able to keep the volume at the end of the titration within approximately the 
same limits (25 to 30 c.c.) in standardizing as in performing the analyses. 

3. Indicator Solution. — The composition of this solution is as follows: 
Sodium citrate 446 grams, sodium nitrite 20 grams, soluble starch 2.5 grams, 
water to 1000 c.c. The starch is first dissolved with the aid of heat in about 
500 c.c. of water. The heating of this solution must be sufficient to accom- 
plish the solution, hence the solution must be boiled for several minutes; if 
starch other than the soluble variety is used, the boiling should continue for 
an hour. The citrate and nitrite are then added and the heating continued 
until all is dissolved. Filter the solution through cotton, wash the filter 
with hot water, allow to cool and make up to i liter. 

Technic. 

Two c.c. of oxalate or citrate plasma are drawn into a dry pipet caHbrated 
to contain 2 + 0.005 c.c. The plasma is run into a 50 c.c. measuring flask 
half full of water, and the pipet is rinsed by drawing the water up into it 
twice. Ten c.c. of the standard silver nitrate solution are added and the 
mixture is diluted to the 50 c.c. mark with water and shaken at intervals 
for several minutes, until coagulation is completed. The addition of a drop 
or two of caprylic alcohol prevents foaming and faciHtates coagulation. The 
solution is now filtered through a dry, chlorid-free filter, the first portion of 
filtrate being passed through, if necessary, a second time to remove turbidity 
completely. 20 c.c. duplicate portions of the filtrate are measured with a 
calibrated pipet into 100 c.c. Erlenmeyer flasks, 4 c.c. of the starch-citrate in- 
dicator solution are added to eac'h, and the standard KI is run in from a buret 
until a permanent blue end-point is obtained. 

Calculation. — This is very simple when standard solutions of the above 
concentration are used. The 20 c.c. of filtrate used for titration represent 
0.8 c.c. of plasma, and the unprecipitated portion of an amount of silver 
nitrate equivalent to 8 mg. of NaCl, or 10 mg. per c.c. of plasma. Each 
c.c. of KI used in the titration is equivalent to i mg. of NaCl per c.c. of 
plasma. Hence the calculation simphfies to 10.15 c.c. of KI = mg. 
NaCl per c.c. of plasma or grams NaCl per liter of plasma. The use of 
10.15 instead of 10 is due to the fact that 0.15 c.c. excess of KI solution is 
required to give the end-point. In this work it is necessary to check the 
accuracy of all pipets, burets, and measuring flasks used by caUb ration.^ 

^ Other methods, or modifications of above, may be found as follows: Harding and 
Mason, Jour. Biol. Chem., 1917, XXXI, 55; Foster, Ibid., 483; Rappleye, Ibid., 1918, 
XXXV, 509; Greenwald, Ibid., 1919, XXXVIII, 439. For studies of the chlorids of the 
plasma see Frothingham, Am. Jour. Med. Sc, 1915, CXLIX, 808; Christian, Ibid., 1916, 
CLI, 630; O'Hare, Arch. Int. Med., 191 6, XVII, 711; Wolferth, Am. Tour. Med. Sc, 191 7, 
CUV, 84; Steinfeld, Arch. Int. Med., 1919, XXIII, 511. 



THE BLOOD 507 

Phosphates. 

These compounds exist in the blood as neutral or alkaline salts of sodium, 
potassium, calcium,^ and magnesium, as well as in organic combinations in 
the red and white cells in the form of lecithin and nuclein. The inorganic 
phosphates are concerned, at least in part, with the solubility of the proteins 
and in maintaining the reaction of the blood through their ''buffer" action. 
Just what variations in the amounts of the organic and inorganic phosphorus 
compounds occur in health and disease is unknown. It is certain that a 
definite phosphorus metabohsm exists and that this is characterized, to a 
great extent, by variations in the normal relationship between the compounds 
of the alkali and alkali-earth groups. Just what these relations are in 
physiologic and pathologic states experiment must determine. That the sys- 
tem retains compounds of phosphorus more energetically than it does any 
other mineral constituents is proven. As the phosphates of the blood and 
of the urine come both from the food and from the breaking down of the nu- 
clein-containing protein material of the system, large variations are possible 
under the influence of many factors. The subject of the metabolism of phos- 
phorus is taken up under The Urine. 

Iron. 

The iron of the blood is found principally in the hemoglobin which con- 
tains about 0.42 per cent, of Fe. It is also found in traces in the plasma, and^ 
according to Hammarsten, in the nuclein compounds. The attempt to esti- 
mate the amount of hemoglobin by determinations of the blood iron have 
proven failures, as no definite relations exist between them. All of the blood 
iron is not in the form of colored compounds and, moreover, some of the de- 
rivatives of hemoglobin are iron-free. Biernacki has shown that the direct 
quantitative estimation of iron yields higher results than could be obtained 
by computation from the percentage of hemoglobin, this finding being con- 
firmed by Jolles and Jelhnek. 

According to Schmidt and Becquerel and Rodier, the amount of iron in 
the blood ranges between 0.056 and 0.058 per cent, while Berman's^ figures 
range from 0.0402 to 0.0559 per cent. Female blood shows a somewhat 
lower value, just as it does for hemoglobin. For the estimation of the 
the iron of the blood, I must refer to other works for the details, as the 
method is too cumbersome for general chnical use. The principle of the method 
is based on the comparison of the colors of a known solution of iron treated with 
ammonium sulphocyanate solution, with that of a solution of blood iron, ob- 

1 See Linzenmeier, Zentralbl. f. Gynak., 1913, XXXVII, 958; and Morley, Surg., Gynec, 
and Obst., 1913, XVII, 276. For studies of the metabolism and methods for determining 
these constituents see the following: For phosphates, Marriott and Haessler, Jour. Biol. 
Chem., 1917, XXXII, 241; Bloor, Ibid., 1918, XXXVI, 33; Feigl. Biochem, Ztschr., 1918,. 
XCII, i; Ibid., 1919, XCIV, 293 and 304. For Calcium see Rowland, Haessler, and Mar- 
riott, Jour. Biol. Chem., 191 6, XXIV, proc. XVIII; Laws and Cowie, Am. Jour. Dis. Child., 
1917, XIII, 236; Lyman, Jour. Biol. Chem., 1917, XXIX, 169; Ibid., XXX, i; Halverson 
and Bergeim, Ibid., XXXII, 159; Halverson, Mohler, and Bergeim, Jour. A. M. A., 1917:. 
LXVIII, 1309; Jour. Biol. Chem., 1917, XXXII, 171; Marriott and Howland, Ibid., 233; 
Cowie and Calhoun, Ibid., 1919, XXXVII, 505. For Potassium, Clausen, Jour. Biol. 
Chem., 1918, XXXVI, 479- 

2 Jour. Biol. Chem., 1918, XXXV, 231. 



5o8 DIAGNOSTIC METHODS 

tained by incinerating blood, fusing the ash with potassium bisulphate, dis- 
solving the fused mass in water, and treating it with sulphocyanate solution. 
The instrument used for the colorimetric tests is called Jolles' ferrometer, the 
amount of iron in i,ooo c.c. of blood being obtained by reference to a table 
accompanying the instrument. If it is desired to obtain the actual percent- 
age by weight, the calculation must include the determination of the specific 
gravity of the blood. Having this latter factor, we may use the equation 
G : V : : TOO : X, in which G represents the specific gravity and V the percent- 
age by volume of iron which is obtained from the table The hemoglobin 

value may be found by use of the formula of von Jaksch, Hb = -, in 

which M equals the percentage of iron by weight. 
(/) Blood Gases. 

The gases existing in the blood are oxygen, carbon dioxide, and nitrogen, 
the latter having httle importance in the body functions, its amount corre- 
sponding to that which would be absorbed by an equal volume of distilled 
water, namely, 1.8 volumes per cent. 

The amount of oxygen and of CO2 varies widely, depending on the arterial 
or venous character of the blood, upon the velocity of the blood flow, upon the 
temperature, amount of exercise, etc. Oxygen occurs principally in chemical 
combination with hemoglobin, but a small amount, about one-fourth per cent., 
is present in solution in the plasma.^ About one-tenth of the CO2 is held in 
solution in the blood, while the remaining nine-tenths is apportioned as 
follows, one-third loosely bound to the alkaUes and hemoglobin of the cor- 
puscles, two-thirds held in chemical combination with the alkalies and pro- 
teins of the plasma. 

The following table made up from the figures of Setschenow, Ludwig 
and Sczelkow, and Hammarsten, will show the relation between these gases. 

Arterial, Venous. 

Oxygen, 21.6% by volume. 6.8% by volume. 

Carbon dioxid, 40.3% by volume. 48.0% by volume. 

Nitrogen, 1.8% by volume. 1.8% by volume. 

These figures are somewhat at variance with those given by Lundsgaard and 
by Harrop. The former states that the maximum oxygen content of normal 
human venous blood is 17.98 volumes per cent., the minimum value is 9.55, 
and the average is 13.6. The difference between the total oxygen-combin- 
ing power of the hemoglobin and the oxygen in the venous blood is termed 
the '^ oxygen unsaturation " of the venous blood, the average figure thus 
being 5.8 volumes per cent. Harrop shows that the oxygen content of the 
arterial blood ranges from 13.89 volumes per cent, to 24.08, with a percentage 
saturation of 94.3 to 100; the oxygen content of venous blood varies from 
10.52 to 17.61 volumes per cent.; the carbon dioxid content of arterial blood 
ranges from 44.58 to 54.69 volumes per cent., while that of venous blood 

^ See Peabody (Jour. Exper. Med., 1913, XVIII, 7) for a discussion of the oxygen con- 
tent in pneumonia; also, Preti, Riforma Med., 1915, XXXI, 432, for variations in blood 
gases in uremia. 



THE BLOOD 509 

varies from 48.27 to 60.43 volumes per cent. These figures vary, of course, 
with conditions which interfere with the proper exchange of gases in the 
lungs and on the influence of the carbon dioxid of the blood on the respira- 
tory center, as well as upon the power of the blood to take up carbon dioxid 
through the agency of the buffer salts of the plasma.^ 
(K) Ferments of the Blood. 

The fact has been well estabhshed by Jacobi that the various organs 
and tissues of the body contain ferments which are proteolytic as far as the 
corresponding tissues are concerned, but are usually inactive when applied to 
the proteins of other organs.^ In other words, these ferments are autolytic, 
but not, as a rule, heterolytic. Much experimental work of former and latter 
years has shown that many metabohc processes, associated with the building 
up and breaking down of various tissue elements, are influenced to a great 
extent by the presence of ferments arising from many sources. 

It is not unreasonable to assume that the blood, like other tissues, contains 
various ferments which have to do with general and special metaboHsm. 
The work of Schonbein on the oxidases of the blood, of Hanriot and, indirectly, 
of Castle and Loevenhart on the lipolytic ferment, of Lepine, Seegen, and 
Spitzer on the glycolytic ferment shows that such properties are resident in 
the blood. The work of Ascoh and Moreschi, Jochman and Miiller, and of 
Stern and Eppenstein has opened up an entirely new field of work on the 
proteolytic properties of the leucocytes. This proteolytic ferment action is 
both autolytic and heterolytic and is influenced to a great extent by the 
checking action of the antiferments, which have been so well studied by Opie. 
Further, it has been shown that differences exist between the proteolytic prop- 
erties of the many varieties of leucocytes and that the ferments are not always 
heterolytic (Mosse). 

It would lead me too far afield to discuss this subject in detail; hence, 
I must be content with reference to the presence of these substances.^ Much 
benefit will be forthcoming from a further study of such properties of the 
blood and of certain constituents of the blood. It is to be recalled that fer- 
ment action may be accountable for the influence of the toxophore and other 
groups of Ehrlich 's complement, but this phase must be discussed later. 
(7) Enumeration of Red and White Cells. 

This section of hematological technic is, perhaps, the most perfected and 
most frequently employed. The red and white cells are usually counted, as 
the enumeration of the platelets has Uttle practical or scientific value at the 

^ See Van Slyke, Jour. Biol. Chem., 1918, XXXIII, 127; Lundsgaard, Ibid., 133; Jour. 
Exper. Med., 1918, XXVII, 179, 199 and 219; 1919, XXX, 147; Harrop, Ibid., 241. 

2 See Menten (Jour. Med. Res., 1919, XL, 433) for a study of general oxidase reactions. 

^ Smithies (Jour. Am. Med. Assn., 191 2, LIX, 539) has called attention to the presence 
in human blood-serum of an agent (probably a ferment) which shows peptid-splitting proper- 
ties. See Caro (Ztschr. f. klin. Med., 1913, LXXVIII, 286), who finds a lipolytic ferment in 
the serum. Also, Neumann, Biochem. Ztschr., 1913, L, 347; Zamorani, Pediatria, 1914, 
XXIII, 401; Porter, Miinch. med. Wchnschr., 1914, LXI, 1775; Resch, Deutsch. Arch. f. klin. 
Med., 1915, CXVIII, 179; Satta, Arch. Ital. Biol., 1915, LXIV, 118; Sagal, Jour. Med. 
Res., 1916, XXXIV, 231; Killian and Myers, Proc. Soc. Exper. Biol, and Med., 1916, 
XIV, 32; Myers and Killian, Jour. Biol. Chem., 1917, XXIX, 179; Sloan, x\m. Jour. 
Physiol., 1917, XLII, 558; De Niord and Schreiner, Arch. Int. Med., 1919, XXIII, 484; 
Burge, Jour. Lab. and Clin. Med., 1919, V, 59; Fujimoto, Am. Jour. Physiol, 1919, L, 208. 



5IO 



DIAGNOSTIC METHODS 



present time and the latter technic is not sufficiently perfected to admit of 
conclusions. 

Various instruments have been introduced for the purpose of counting 
the corpuscles in a given volume of blood. Most of them are based on the 
principle that a layer of diluted blood, of a certain depth, covering a certain 
known space, shows a definite number of corpuscles for every drop of blood 
used. The general method of making the count consists in diluting the 
fresh blood in definite proportions with some indifferent fluid and counting, 
under the microscope, the number of cells in a drop of this diluted blood, 
which is contained in a small glass cell on the floor of which is ruled a series 
of micrometer squares of known dimensions. The cubic contents of the 
cell and the degree of dilution of the blood being known, the number of cor- 
puscles counted in any given number of squares of the ruled area may be 
taken as a basis for calculating the total number of cells in a cmm. of blood. 
Strong and Seligmann dispense with a special counting chamber and enumerate 
the cells in a definite quantity of blood diluted in exact proportions with a 
diluent stain and mounted as a permanent specimen. Einhorn and Laporte 
use a somewhat similar method and arrive at very good comparative results. 

The normal number of red cells in the adult male is, approximately, 
5,000,000 per cmm. of blood, while in the female it is somewhat lower, namely, 
4,500,000. Marked variations in this figure are observed in pathological 
conditions and will be discussed in a later section along with the treatment 
of the physiological factors which influence the number and appearance of 
these erythrocytes. In the normal adult the number of white cells varies 
between 5,000 and 10,000 the average being 7,500. This figure is subject to both 
physiologic and pathologic influences to a greater extent than are the red cells. 

Many instruments, such as those of Hayem, Gowers, Malassez, Thoma- 
Zeiss, Alferow, and Durham, have been introduced, but the most universally 
used and the best adapted for such investigation is, in the writer's opinion, 
that of Thoma-Zeiss. This combines certain modifications of the mixing 
pipet of Malassez, the counting chamber of Hayem, and the micrometer 
rulings of Gowers. It gives us, therefore, a most complete instrument for such 
work. 




Fig. 130. — Thoma-Zeiss counting chamber. 

Hemocytometer of Thoma-Zeiss. 

The blood counter, originated by Thoma and constructed by Zeiss, is 
all that could be desired. Leitz, Reichert, and other makers of optical 



THE BLOOD 



511 




goods have introduced similar types of counters, but our experience with 
them has not been so good. So much depends upon the accuracy with which 
.pipets are graduated and upon the trueness of the rulings of the counting 
chamber that we recommend the general use of the Zeiss instrument. 

This instrument consists of a pipet for mixing the blood to a certain 
dilution, a counting chamber by means of which a layer of known depth 
and area is obtained, and a special cover-glass for the chamber. 

Pipets. 

The original form of the Thoma apparatus included but one pipet this 
being used for the dilution when both red and white cells were to be 
counted. Experience has shown, however, that the 
dilution given by this pipet is, in most cases, too great 
to permit of accurate counting of both types of cell.^ 
A modification has, therefore, been introduced to allow 
of greater accuracy by giving a lower dilution and by 
furnishing a larger number of cells in the counting 
chamber. 

Er3rthrocytometer. 

This is the original pipet of Thoma. It consists of 
a graduated capillary tube (A) opening into a dilatation 
(B), at the opposite end of which is a shorter, glass tube 
(C), graduated with a line marked loi and to which is 
attached a rubber tube with an ivory mouth-piece. 
This pipet is so graduated that the capacity of the 
ampulla (B) is exactly 100 times that of the capillary 
tube from its point to the line marked i and 200 times 
that from the point to the hne marked 0.5. Other 
lines, both below and above this latter point, are cali- 
brated on the tube, each line representing one-tenth 
of the capacity of the capillary. In some of the 
pipets, especially that of the Miescher hemoglobi- 
nometer, two smaller marks each representing one one- 
hundredth of the length of the tube are calibrated on 
each side of the major divisions. By this means the 
dilution of the blood may be definitely known if the 
tube be not filled exactly to the point described. Those 
pipets with obtuse ends are much to be preferred to 
those with the more acute angles. In the ampulla is a 
small glass bead which is of service in properly 
mixing the blood with the diluting fluid. This pipet may be used in counting 
both the red and white cells and is all that is absolutely necessary when one 
uses the Zappert or Tiirk counting chamber. In general work, however, 
it is used only in the count of the red cells as the extreme dilution given is 
better adapted to this estimation than it is to the counting of the white cells. 
^ See Ellermann, Deutsch. Arch. f. klin. Med., 1913, CIX, 378. 



\ 



m 

III 

ri 



Fig. 131. — Diluting 
pipets: Erythrocytom- 
eter; leucocytometer 
with Croy's attach- 
ment. 



512 



DIAGNOSTIC METHODS 



Leucocytometer. 

This is a graduated capillary tube similar to the erythrocytometer in 
construction but having a larger lumen, which will permit of lower dilutions. 
It is graduated into ten divisions, with the marks 0.5 and i representing 
these measures of the total capacity of the capillary. Above the ampulla is 
a graduation, 11, which is so calibrated that blood, drawn to the mark i and 
diluted to 11, gives a dilution of 10, while if drawn to the point 0.5 the 
dilution is 20. Those forms of this pipet which have the lower end tapering 
to a fine point giving a gradually decreasing lumen, are much to be pre- 
ferred to the older models. As the caliber of this instrument is relatively 
large, the student is cautioned against using too great suction in making 
the dilution, and also against placing too large a drop on the counting 
surface.^ 

The Coimting Chamber. 

''This consists of a heavy glass slide. A, on which is cemented a thick 
glass ring, B, the surface of which is highly polished. This ring surrounds 




Fig. 132. — Ruled surface of Thoma-Zeiss counting chamber. {Da Costa.) 



a circular table of glass, D, the height of which is just o.i mm. less than 

that of the surrounding ring. Between this glass table and the inner edge 

of the ring is a small ditch, C, to catch the drop which may run off from 

the table and to prevent its flowing up between the ring and the cover-glass 

on the other side of the ditch. On the central glass table cross at right 

angles 21 parallel lines, equidistant, and between the extremes of which is 

exactly i mm. Hence we have an area of i square millimeter divided 

into four hundred small equal squares. Through each fifth row of squares 

^ My colleague, Dr. C. C. Croy, has devised a most excellent and useful pipet attach- 
ment (see above cut), which prevents leakage of the contents during transportation. This 
consists of a strong rubber band with two disks of any kind of thin metal folded on the 
rubber in such a way that the ends of the pipet come in contact with the rubber and press 
against the metal disks. See, also, Saxon and Drummond, Jour. Am. Med. Assn., 1915, 
LXV, 1 182. 



THE BLOOD 513 

is ruled an extra line, which is not a boundary but is merely an aid in 
keeping one's position in the ruled area. Indicated, not bounded, by 
these extra Hnes, the square millimeter is divided into 16 units of 25 small 
squares each" (Emerson). 

As the ruled surface of one square milUmeter is divided into 400 small 
squares, each small square has an area of J-400 square mm. The height of 
the column of blood being Ko mm., the cubic contents of each small square 
is Kooo cmm. 

This counting chamber of Thoma does not permit of the counting of a 
sufficiently large number of leucocytes, especially when the dilution has been 
made in the same degree as is used for the enumeration of the red cells. In 
order to overcome this difficultv and also to give a larger ruled area in which 




Fig. 133. — Turk's ruling of the counting chamber. 



the leucocytes may be counted, Zappert has modified the original ruling in 
such a way that a counting surface of 9 sq. mm. is afforded. This modi- 
fication has been improved by Ewing and by Tiirk in such a way that the 
four large corner squares, each of i sq. mm., are subdivded into 16 smaller 
squares, each of which is equal in area to the total 25 smallest squares of the 
Thoma chamber. This latter modification is much the best and is used 
exclusively by the writer. Its advantage in counting both red and white 
cells will be appreciated when the student compares it with the older chamber. 
The sixteen central squares are used in counting the erythrocytes, while the 
entire area may be used in the enumeration of the leucocytes. Simon has 
recently introduced a different modification of the Thoma ruUng, which is 
extremely simple and should prove very satisfactory. Other forms have 
been introduced by Biirker, Gorjajew and Pappenheim and, more recently, 
by Bass. AU give excellent results. 

Before use the counting chamber should be w^ell washed with water and 
carefuUy dried. Precautions should be taken to see that no hnt is left on 
the surface of the glass ring and that no alcohol or ether are used in the clean- 
ing process, as these substances loosen the cement with w^hich the glass table 
is fastened to the shde. 
33 



514 DIAGNOSTIC METHODS 

The Cover-glass. 

This is made of heavy poHshed glass with accurately planed surfaces. 
The ordinary cover-glasses should never be employed with the counting- 
chamber as they are often uneven in surface and do not fit tightly to the slide. 
Moreover, these ordinary cover slips are so thin that the capillarity of the 
drop of blood may bend them down to some extent. The cover-glass must be 
as carefully cleaned and dried as is the chamber. 

Diluting Fluids. 

In order that the blood may be properly examined, it must be diluted 
with a solution which will at the same time prevent coagulation and hemolysis 
and will preserve the corpuscles intact. There are numerous formulae for 
such solutions and the choice is largely a matter of experience. Much will 
depend on whether the red and white cells are both to be counted at the same 
dilution or whether two different pipets are used in making the dilutions. 
Personally the writer prefers the use of two pipets using different diluents, but 
other workers use one pipet and one diluent. The red cells may be destroyed 
by certain fluids leaving the white cells intact or the white cells may be colored 
by the same diluent used in counting the reds. 

Hayem's Solution. 

This solution preserves the red cells permanently and permits the corpus- 
cles to settle slowly, thus furnishing an even distribution of the cells. More- 
over, it will keep almost indefinitely and does not permit of the development 
of yeast spores which so readily multiply in many of the other diluents. The 
writer can recommend this diluent as the most generally useful of the prepara- 
tions advised. It is made up as follows: 

Mercuric chlorid, 0.500 gram. 

Sodium sulphate, 5.000 grams. 

Sodium chlorid, i.ooo gram. 

Distilled water, 200.000 c.c. 

Owing to the presence of mercuric chlorid, this fluid cannot be mixed 
with an anihne coloring substance to stain the leucocytes and is, therefore, 
not applicable to the combined counting of the red and white cells. ^ 

Toisson^s Fluid. 

Sodium chlorid, i.ooo gram. 

Sodium sulphate, 8.000 grams. 

Neutral glycerin, 30.000 c.c. 

Distilled water, 160.000 c.c. 
Methyl violet 5 B., 0.025 gram. 

The addition of methyl violet serves to color the leucocytes and permits 
of their recognition along with the erythrocytes. Occasionally this fluid 

1 Jorgensen (Ztschr. f. klin. Med., 1914, LXXX, 21) believes the results with this solu- 
tion are much improved by diminishing the mercuric chlorid content to o.i gram (one-half 
part per liter). 



THE BLOOD 515 

hemolyzes the red cells and thus invalidates the count. Moreover, it easily 
becomes infected with yeast spores which develop profusely in it. For this 
reason it is advisable to filter the fluid before use, each filtration, however, 
weakening it, so that it becomes after a time useless. 

Other diluents, such as the solutions of Pacini, Lowit, Petrone, Acquisto, 
Edington, and Callison have been used, but the more generally apphcable 
ones are those mentioned above. Toisson's fluid is particularly useful when 
the count of reds and whites is to be made in the same specimen. The color- 
ing of the leucocytes is not necessary for their recognition, but it is a conven- 
ience to one who is not making blood counts frequently. Hayem's solution 
is the very best diluent at our command for general purposes. 

If it is desired to count the white cells alone, and this is always wise, a 
I per cent, solution of acetic acid, to which is added gentian violet to bring 
out the white cells a little more clearly, may be used. This solution destroys 
the red cells and thus gives only the white cells in the preparation. For this 
reason the addition of the gentian violet is unnecessary. Yeast cells develop 
in this solution with more or less readiness, hence one should employ only 
freshly made solutions, as these yeast cells resemble, to some extent, mono- 
nuclear leucocytes and may introduce an error into the count. ^ 

Method of Counting the Corpuscles. 

With this process of counting the cells, whether red or white, there are five 
steps to be taken : 

1. Obtaining the blood. 

2. Diluting and mixing the blood. 

3. Filling the counting chamber. 

4. Counting the cells. 

5. Cleaning the apparatus. 

Erythrocj^es. 
(i) Obtaining the Blood. 

As previously stated, the blood may be drawn from a puncture of the 
ear or finger. Personally, the writer always uses the ear, unless some vahd 
reason exists for not doing so. As soon as a good-sized drop appears, which 
is obtained without pressure or constriction, the tip of the pipet is placed in 
the drop and is supported by a finger of the left hand, which holds the ear in 
position. The blood is drawn by suction to the mark 0.5, in cases in which 
anemia is not suspected, or to the mark i in such cases, as a routine the former 
mark being preferable. As the student will find, some practice is needed to 
stop the column of blood exactly at the point desired. If the blood be drawn 
a little too far, the tip of the pipet may be rubbed with the finger or the excess 
may be shaken down by tapping the tip against a towel or, perferably, the 
blood may be drawn to the next mark and the necessary correction made in 
the dilution. Unless this error can be corrected, the pipet must be cleaned or 

^ Seilin (Jour. A. M. A., 1916, LXVII, 1387) calls attention to the fact that a trace of - 
copper sulphate added to this solution prevents the development of molds and yeasts. 



5l6 DIAGNOSTIC METHODS 

a second one used. In any event, if the blood be not accurately measured, 
we must reject the determination. Moreover, the work should be done 
rapidly to prevent coagulation of the blood. Hence, too much time should 
not be spent in adjusting the column of blood. ^ 

(2) Diluting and Mixing the Blood. 

As soon as the column of blood is adjusted at the desired height of the 
capillary, the tip of the pipet is carefully wiped with the fingers to remove 
any adherent blood and is immediately dipped into the diluting fluid in such 
a way that no portion of th^ blood is lost. Tiirk recommends closing the end 
of the pipet with the finger and exerting slight suction on the closed tube in 
order to prevent any loss on immersion of the pipet. I have never seen it 
necessary to use this measure, providing ordinary care is employed in applying 
the suction as soon as the pipet touches the diluent. The diluting fluid 
should stand ready in a small dish or bottle and should be carefully examined 
before use to see that no flocculi or spores are present. 

The diluent should be at once drawn into the capillary by suction. The 
fluid rises slowly in the tube, the pipet which is held vertically being rotated 
between the finger and thumb of the left hand as the fluid rises. By this 
rotation, the diluting fluid is mixed with the blood at once and bubbles of air, 
which often cling to the inside of the tube, are avoided. The glass bead in the 
ampulla serves the purpose of thoroughly mixing the blood and diluent. As 
the column approaches the mark loi on the upper end of the pipet, care 
should be taken that the aspiration is not too strong. While the error, intro- 
duced by drawing the diluted blood beyond this mark, is not as great as it is 
in drawing the undiluted blood to the mark 0.5, yet both should be avoided. 
When this upper mark is reached, withdraw the pipet from the diluent, close 
the tip with the finger, bend the rubber tubing down over the other end, and 
close this with the thumb. Some prefer to remove the rubber tubing at this 
juncture, but this is not at all necessary. Shake the pipet vigorously for at 
least one minute to insure a thoroughly uniform mixing of the contents. If 
for any reason the count is not to be made at once or if it be desired to carry 
the blood to the laboratory for examination, the rubber tubing may be re- 
moved and the ends of the pipet closed by means of a rubber band. Natu- 
rally, before the mixture can be used it must be again thoroughly shaken. If 
the examination is to be done at once, proceed as follows. Blow three or 
four drops from the pipet in order to remove the column of fluid which has 
remained in the capillary and has not mixed with the blood. If Toisson 's 
fluid has been used as the diluent, it is better, before blowing out the drops, 
to allow the pipet to lie horizontally for 10 to 15 minutes in order to permit of 
the staining of the leucocytes. (__ 

(3) Filling the Counting Chamber. 

As a rule, it is better, unless Toisson's fluid has been the diluent, to fill 
the counting chamber at once as errors may creep in by allowing the pipet 

^ See Maddox (Jour. Am. Med. Assn., 1913, LX, 663) for the description of a new device 
aiding in filling the pipets. 



THE BLOOD 517 

to stand, even though the later mixing may be thorough. A small drop, the 
size of which can be learned only by experience, is blown onto the center 
of the ruled area of the counting chamber. The drop should not be so large 
as to run over into the moat, but should be large enough practically to cover 
the glass table. 

Adjust the cover-glass at once. This is a point in the technic which 
requires considerable practice and must be mastered before accurate results 
can be obtained. Emerson's advice on this point is admirable, ''grasp the 
cover-glass by two diagonal corners, place a third corner against the shde 
with the edge of the glass ring as a fulcrum, and hold it in that position by a 
finger of the left hand. By now raising the finger the cover is rotated onto 
the drop rapidly and also in such a way that no air-bubble is left." Breath- 
ing upon the cover-glass before it is adjusted is often serviceable in making 
this preparation. If there has been no dust on the slide or cover-glass and 
they are perfectly clean, the student will observe, providing the adjustment 
has been properly made, a beautiful band of colors known as Newton's rings, 
which are due to a phenomenon of interference of Hght. If these rings do not 
appear, they may often be brought out by firm pressure on the edges of the 
cover-glass. If they are not persistent, after the pressure has been removed, 
the adjustment of the two poHshed surfaces must be assumed to be imperfect 
and the preparation rejected.^ It happens at times that these rings are 
difficult to obtain and some workers state that they are not necessary for an 
accurate count. The writer prefers, however, to reject those sHdes which do 
not show these diffraction rings than to run the risk of including a possible 
error. These rings may best be seen by holding the shde on a level with the 
eyes in such a way that the light is totally reflected from the surface of the 
eover-glass. After the shde is properly adjusted allow it to stand for three 
or four minutes before proceeding with the next step of the determination, in 
order to insure the proper setthng of the corpuscles upon the counting table. 

(4) Coimting the Cells. 

Before any count of the cells is attempted, the entire surface covered by 
the blood must be examined with the low-power lens to ascertain whether the 
distribution of the cells is uniform throughout. If not, the slide should be 
rejected, even though the points mentioned previously have obtained. It is 
much better to stop proceedings at this point than to attempt to equahze an 
uneven distribution by a larger counting area. If the specimen proves satis- 
factory the count may then be undertaken. The lenses best suited to this 
purpose are Bausch & Lomb %, Zeiss D, Leitz 6 or 7, and Reichert }i. As 
the student becomes accustomed to cell-counting he may use a lower objec- 
tive. This has the advantage of bringing the entire field of one sq. mm. into 
focus. A mechanical stage is of some convenience in making the count, but 

^ Eustis (Jour. Am. Med. Assn., 1913, LXI, 1984) advises the following: Grasp the 
cover-glass between thumb and index-finger of right hand. Hold the slide firmly on the 
table with the left hand and rapidly shde the cover-glass across the counting chamber, 
moving the glass in a perfectly horizontal manner. See, also, Hartz, New York Med. Jour., 
1915, CL, 612; Lyon (Jour. A. M. A., 191 7, LXVIII, 709) calls attention to possible 
inaccuracies in the size of the counting chamber and pipets. 



5l8 DIAGNOSTIC METHODS 

the fingers answer quite as well after some practice in manipulating 
the shde. 

The unit of counting surface is a matter of individual preference. Sahli 
recommends a unit of four small squares, Grawitz and Simon use sixteen of 
these squares, while Cabot advises the use of 36. In common with Tiirk, 
Ewing, Da Costa, Wood, and Emerson, the writer prefers the units of 25 small 
squares, as this is the unit of the ruhng and the calculation is much simpler 
than with the other units. 

In order to simplify the process of counting, some routine method must 
be used. These methods depend on the worker, but the usual procedure in 
the writer's laboratory is as follows: Adjust the slide upon the table of the 
microscope so that the upper left-hand corner of the central ruled area of 16 
large squares of the Tiirk chamber is brought into the field in such a manner 
that one may count the cells in the small squares from left to right. If the 
low-power objective is used, the complete group of 16 units will lie within the 
field of observation and may be easily examined. With the higher power, 
which the student may be forced to use in his earlier work, the optical field is 
necessarily limited. Count the total number of cells lying within the 25 
small squares of the single unit. In doing this count the upper row of the 
unit from left to right, drop down a row, and count the cells from right to left 
and so on until the cells in the whole unit have been counted. The accom- 
panying cut will indicate the method to be followed. In making this count, 
cells which touch the right hand or lower boundaries of the unit are disre- 
garded, while those which touch the upper and left-hand hne are included in 
the count of that square. After counting the cells in the upper left-hand unit, 
count those in the remaining fifteen units, thus covering a field of 16 units of 
25 small squares each, making a total of 400 small squares counted. Tiirk 
recommends the counting of eight units as a minimum, Emerson advises 
the counting of the four corner units in each of two separate preparations, 
while Da Costa and others count the cells in four groups of units from above 
downward and repeat with four units not adjacent. It seems to the writer 
that the error is less the larger the area covered, and he, therefore, advises 
the beginner to make the total count of the sixteen units, although it is not 
supposed that one will try to cover up defects in technic by this larger count- 
ing area. A variation of more than 25 cells in the counts of the various 
units should be taken as evidence that the distribution is not perfect. In 
such cases the count would better be rejected on the ground of inaccuracy. 
After the technic is mastered and the worker has discovered just exactly 
wherein his error lies, a count of 8 or 4 units will suffice. It is advisable 
where accurate scientific results are indispensable, to clean the slide and make 
a second count with a fresh drop of blood so that one may have a check on 
his work. For clinical purposes, however, the count of four units will ordi- 
narily be sufficient. 

In ordinary counting it is not necessary to differentiate the red cells from 
the leucocytes, as the error thus introduced is small and may be disregarded. 
In cases, however, which show large leucocyte values, this error will be quite 



THE BLOOD 



519 



appreciable and must be overcome by the following method. All of the cells 
observed may be counted as erythrocytes and the reduction made for the num- 
ber of leucocytes as obtained by the special leucocyte count to be described 
later. In the use of Hayem's diluting fluid, the leucocytes are not colored, 
while the erythrocytes retain their normal yellow color. The leucocytes ap- 
pear, by good illumination of the field, of a bluish tone, are somewhat larger 
than the red cells, and are characterized by a sharper border. These facts will 
enable one after some experience to distinguish the white from the red cells. 




Fig. 134. — Plan of Counting the Cells, {DaCosta.) 
The small squares are examined in the order indicated by the arrow. 



Toisson's fluid stains the leucocytes blue, and may, therefore, be used to 
outhne these cells. As a rule, it is better to learn to recognize the leucocytes 
by differences in refraction than to rely on the staining qualities of these cells. 
In some cases the methyl violet of the diluent colors some of the red cells so 
that they cannot be easily distinguished from the white ones. 

The number of cells in the blo'od is invariably reported as the number 
contained in a cubic miUimeter of blood. In making the calciflation of this 
number it is necessary to know the number of units counted, the number 
of cells in these units, the area of each small square of the unit, and the degree 
of dilution of the blood. Thus, if 16 units, each of 25 squares, have been 
counted, the total number of small squares is 400, each having a cubic area 
of MjOOO cmm. The total volume of the units counted is, therefore, Ho cmm. 
It is evident that the number of cells in i cmm. of blood is 10 times that in 
the area counted over if the blood were undiluted. But the count is always 
made with diluted blood and we must, therefore, take this factor into account. 
With a dilution of 100 multiply the number of cells in i cmm. by 100, and 
with a dilution of 200 multiply by this factor. Thus, if 2,500 cells were 
counted in the 400 small squares and the cubic contents of the units gone over 



520 DIAGNOSTIC METHODS 

was }io cmm. then i cmm. of diluted blood would contain 25,000 cells. As 
the blood was diluted 200 times, the total number of cells in one cmm. of 
undiluted blood is 5,000,000. A very simple way of remembering this calcu- 
lation is to multiply the number of cells counted in the total area of 400 
small squares by 1,000 if the dilution was 100, and by 2,000 if the dilution 
was 200. 

If the total area of 16 units be not counted, the method of calculation is 
the same but the factors are variable. This method goes as follows: Multi- 
ply the number of cells counted by the degree of dilution and this result by the 
cubic contents of each small square (4,000). Divide this result by the num- 
ber of small squares counted. Thus, to calculate the number of cells in a 
cmm. of blood when 100 small squares (4 units) were counted at a dilution of 

200, the count being 625 cells, the equation is as follows: 

= 5,000,000. 

Leucocytes. 

In counting the leucocytes much depends on the sort of ruled slide at 
the disposal of the worker. With the old Thoma chamber at least five 
different drops must be examined in order that a sufl&ciently large number 
of leucocytes may be counted, while with the Tiirk cell a counting area of 
9 sq. mm. is afforded for each drop. The more leucocytes counted so much 
the less is the error. It is usually sufficient to count the white cells in a 
single drop, using the Tiirk chamber, but for scientific purposes three or 
even four drops would better be examined. 

If it is desired to count the leucocytes in the same specimen as the red cells 
the procedure is as follows : Prepare the drop of blood exactly as described for 
counting the red cells, using Toisson's fluid as the diluent and the erythrocy- 
tometer as the diluting pipet. After the reds have been counted, enumerate 
the whites in the entire ruled area of this chamber. In this process the leuco- 
cytes will be found to be stained a faint blue. It is often advisable, in case of 
low leucocyte counts, to repeat this process with a second drop of blood. The 
calculation by this method is very simple. As the entire ruled area of the 
Tiirk chamber covers a surface of 9 sq. mm., each equal to the central area 
used in counting the red cells, we have the equivalent of 3,600 small squares 
in the ruled surface. Multiply this figure by the number of drops used to 
obtain the total number of small squares covered by the count. Thus, if 54 
leucocytes were observed in two drops (7,200 small squares) and the dilution 

54 X 200 X 4000 ^ „ 

was 200, then we have the equation, = 6,000 cells. 

' ^ ' 7200 ' 

A second method of calculating the number is to consider each sq. mm. 

of the surface of the Tiirk chamber as a unit. If then, the number of cells 

counted in two drops (18 units) be 54, we divide this number by the number 

of units counted, 18, and multiply the result by 10 (the cubic contents of each 

unit) and then by the dilution. Thus, 5 = 6,000. 

lo 



THE BLOOD 52 I 

It is usually preferable in counting the leucocytes to use the special leuco- 
cytometer previously described, as this gives a smaller dilution, and conse- 
quently a larger number of leucocytes to the counting surface. In using this 
pipet, the blood is drawn to the mark i and the diluent, i per cent, acetic 
acid, added to the mark ii. This diluent destroys the red cells and brings 
out the leucocytes clearly. The dilution of the blood will thus be lo. If a 
large increase in the number of leucocytes is anticipated, it is better to use 
a dilution of 20, drawing the blood to the mark 0.5, as a routine the dilution 
of 10 being, however, preferred. 

In counting the white cells with a dilution of 10, using the Tiirk chamber, 
it is not as a rule necessary to go over the entire counting field, but more ac- 
curate results will obtain if this be done. A count of at least 250 cells is ad- 
visable, while one of 1,000 is more to be preferred in scientific work. The 
method of calculation is the same as given above. Thus if 540 cells were 
counted in the 9 sq. mm. of the ruled surface, the dilution being 10, we have 

1540 X 10 X 10 ^ -rr 1 11 1 11 

= 6,000. It we nave not used the total 9 sq. mm., but have 

used 4 or any other number which may be considered sufficient by the 
worker, the divisor in this division will be the number of units counted. 

If only a Thoma chamber be at hand for counting the cells, two methods 
are available. By the first, several drops may be gone over counting the total 
number of cells in the ruled sq. mm. of the cell. In the second method 
we find the cubical contents of each visual field and then count the leucocytes 
in many of such fields. The method of computing these factors is given by 
StengeP to whose work the author refers the reader who may not have access 
to a Zappert or a Tiirk chamber. 

In making this leucocyte count great care must be exercised to have 
the diluent fresh and free from yeast spores, which so freely develop in such 
mixtures. If this factor be observed, all the cells seen may be counted as 
leucocytes, but occasionally nucleated red cells may be confusing, especially 
if these be present in large numbers. The physiological condition of the 
patient should always be considered in making a report on a leucocyte 
count, as such factors as digestion and exercise influence this count to a 
great extent. 

The normal error in making a leucocyte count, with a count of 200 and 
more leucocytes, is about 5 per cent., while in the case of a red count it should 
not be over 3 per cent. Careful work with special attention to all the details 
mentioned will often reduce this error to a lower figure. Naturally, the error 
in counting the leucocytes will be much reduced by using the Tiirk chamber 
and giving the blood a dilution of 10. There are certain errors due to faulty 
construction both of the pipet and of the counting chamber which remain 
constant in the same apparatus Hence it is wise to procure the very best 
equipment possible and to test the different portions of the pipet for such 
errors. In this way a very appreciable difference may be obviated. Not all 

^ Twentieth Century Practice of Medicine, New York, 1896, VII, 271. 



522 DIAGNOSTIC METHODS 

of these blood counters show such variations, but some do, and it is, therefore, 
a matter of moment to know your tools. 

(5) Cleaning the Apparatus. 

This is the last step in the technic of making a blood count. While 
perhaps not as important as some of the other steps, yet if not properly carried 
out it will introduce errors which may prove very annoying. It is readily 
seen that an unclean pipet or counting chamber will interfere with the proper 
manipulations as described above. 

After the cover-glass has been removed from the slide, wash out the 
chamber with distilled water and dry thoroughly by means of a clean piece of 
linen. Place the slide in its proper receptacle, so that it may be conveniently 
found when desired. This may seem a small point, but the author has seen 




6 



Fig. 135. — Cross-section of Durham's blood pipet. {Da Costa.) 
T, Glass tube; n, rubber nipple; p, lateral perforation in nipple; c, cork in which a 

capillary pipet is fitted. 

too many slides lost by carelessness in putting them away. The cover-glass is 
rubbed clean and dry and put in the case with the slide. 

Wash out the pipet with water until all of the blood is gone. In some 
cases fine clots will be observed sticking to the side of the tube. Under such 
conditions remove the adherent blood by means of a fine wire or, if this is 
not effective, draw a little strong potassium hydrate solution into the capil- 
lary and allow it to act on the clot. After the tube is apparently clean, wash 
it out with alcohol and ether. Blow a stream of air through the tube by 
means of compressed air or the suction pump. Be certain that the apparatus 
is perfectly dry and clean, and that the glass bead in the ampulla is freely 
movable as the tube is shaken, before putting the pipet in its case. 

Durham's Hemocjrtometer. 

Recently Durham has introduced a modification of the older instruments 
for blood-counting. This embodies the principles of the various methods, 
but substitutes a self-measuring capillary pipet for the suction pipet of the 
Thoma apparatus, and special mixing vessels for the dilution of the blood. 
This device makes it possible for one inexperienced in blood-counting accu- 
rately to measure the blood and diluting fluid and thus eliminates the error 
possible with the older pipet. The direct count is made in the counting 
chamber of Thoma. This capillary of Durham is more easily cleaned than 
that of Thoma, thus giving an advantage in cases where several blood 
examinations are to be made in a limited time. While this method of diluting 
the blood has the advantages above mentioned, the writer has never been 
able to convince himself that the use of this apparatus gives the student any 
better results than he obtains with the Thoma instrument. After some 



THE BLOOD 



523 




experience, and this is needed in any method, the dilution of the blood with 
the Thoma pipet is quite as easily made as with the Durham modification, 
and the results of the count give quite as close checks as do those with the 
newer method. 

Oliver's Hemocytometer. 

This instrument was intended to furnish a more accurate method of count- 
ing the red cells than was given by the older instruments. The method is 
based on a principle entirely different from that of the older instruments and 
does not afford an actual count of the cells. If blood be 
diluted with a fluid, which preserves the corpuscles, in a 
rectangular test vessel composed of longitudinally stri- 
ated glass, each striation of the glass will act as a lens 
projecting an image of a candle flame viewed through the 
suspension of opaque particles of the blood, providing 
the suspension is of a sufficient dilution to permit of the 
almost unobstructed passage of the rays of light. x\t the 
proper dilution, these images of the candle flame will 
form a bright streak horizontally across the tube. Ex- 
periments have shown that the development of this bright 
line, on dilution of the blood with Hayem's solution, is an 

accurate measure of the percentage of red cells in the Fig. 136.— Oliver's 
, ™, ,., . ,, ,, ,. ,. hemocytometer. 

Specimen examined. 1 he dilution 01 the blood is made in {Greene.) 

a rectangular glass cylinder by means of a capillary pipet, 

which is w^ashed out with Hayem's solution. The cylinder is graduated into 

divisions from 10 to 120, each division representing 50,000 red corpuscles. 

As this instrument has little clinical value, owing to the fact that the 
error is very great in cases in which the blood is diseased, I refer the reader 
to other works for a description of the method in detail. In the study of the 
physiologic variations of the red cells, this method affords very accurate re- 
sults, giving figures which would be lost wdth the Thoma instrument. It must 
be remembered that, in this method, as in many others the personal equation 
plays a large role and may account for serious error which is not found with 
the Thoma instrument. Emerson has shown that the variations in the re- 
sults may be as much as 2,000,000 cells when this instrument is compared 
with the Thoma in counting the blood in primary anemia. Baumgarten has 
proven that variations in the size of the cell as well as deformities of the cell 
will introduce a serious error into this method. Ellermann and Erlandsen^ 
as well as Dunger^ have recently introduced new methods, for making ac- 
curate counts of the white cells, which may prove very advantageous. 

Cotinting of the Blood-platelets. 

The technic of counting these cellular elements has been imperfect and the 
results variable. The methods advanced are both direct and indirect. The 
technic of Determann and of Brodie and Russell belong to the latter class, 

1 Deutsch. Arch. f. klin. Med., 1910, XCVIII, 245. 

2 Miinch. med. Wchnschr.. 1911, LVIII, 1131. 



524 DIAGNOSTIC METHODS 

while the method of Helber is a direct one. Recently Wright and Kinnicutt^ 
have introduced a method which is simple, exact and reliable. The technic 
is as follows: The blood is diluted i : looby means of the pipet used for count- 
ing the red cells and the counting is done in the Thoma-Zeiss counting-cham- 
ber, using all the precautions previously discussed. The specially thin 
cover-glass of Zeiss, with central excavation, is used to render the platelets 
clearly visible. The diluting fluid consists of 2 parts of a i : 300 aqueous solu- 
tion of "brilliant cresyl blue" and 3 parts of a i :i4oo aqueous solution of 
potassium cyanid. These two solutions should be fairly fresh, kept separate, 
mixed and filtered just before taking the blood. After the counting chamber 
is filled, it is left at rest for 10 to 15 minutes in order to allow the blood- 
platelets to settle to the bottom of the chamber and be more easily and 
accurately counted. The platelets appear as sharply outlined, round, oval 
or elongated lilac-colored bodies, some of which form a part of small spheres 
or globules of hyaline substance. The red cells are decolorized and appear as 
''shadows," while the nuclei of the leucocytes are stained a dark blue and their 
protoplasm light blue. This method shows a normal platelet count of 225,- 
000 to 350,000 per cu. mm. No constant relations seem to obtain between the 
variations in the number of platelets and of the leucocytes. According to 
Determann, the ratio between the red cells and the blood plates is, on the 
average, 22 :i. The pathologic variations in the number of these cellular 
elements will be discussed in detail in a later section. 

III. Morphology of the Blood 

Before any examination which is concerned with the study of the morpho- 
logical characteristics of the blood can be made, it is essential that all of the 
glassware which comes in contact with the blood should be absolutely clean 
and dry. The glass slides, as they come from the shops, are often coated 
with substances which are removable with difl&culty. Moreover, these 
slides are not in all cases perfectly level on both surfaces. It will need but 
one experience with an uneven slide to convince the worker that it is a loss of 
time to attempt the use of such slightly convex or concave slides. The cover- 
glasses should be of the very best quality of glass, should be as thin as possible 
(number o), and three-fourths inch square. The seven-eighths inch square 
covers as also the larger rectangular ones are not as desirable for blood work, 
especially in the examination of fresh specimens. 

The slides and covers should be cleaned with soap and water followed by 
water and alcohol. In some cases it is necessary to soak them in concentrated 
hydrochloric acid for some hours and then wash with water, alcohol, and 
ether, or the ordinary acid-alcohol may be used. After being cleaned they 
should be kept either in 95 per cent, alcohol or, preferably, polished with a 
clean linen cloth or a piece of tissue-paper and kept in dust-proof receptacles. 
It is a wise precaution invariably to polish the slides and covers before use, 
as dust particles are prone to collect even under the best conditions. As a 

^ Jour. Am. Med. Assn., 1911, LVI, 1457. See, also, Fonio, Deutsch. Ztschr. f. Chir., 
1912, CXVII, 176. 



PLATE XVII. 




Kathar 



,t\<2' 



Fresh Normal Blood. (Zeiss Ocular 4, Objective DD.) 



THE BLOOD 525 

rule, it is better to use only new cover-glasses and not attempt to clean them 
after use. The slides may, however, be cleaned by boiling with a strong alkali 
solution, washing with hot water acidihed with hydrochloric acid, then with 
hot water, alcohol, and ether. 

After the cover-glasses have been polished it is the best practice to handle 
them only with forceps, as moisture is almost certain to collect on them if the 
hngers be used. This is not only better technic, but more rapid work may 
be done with their use. Two kinds of forceps are necessary in such work. 
The first is one for holding the cover firmly, being found as the locking forceps 
of Ehrlich or the cross-point forceps, while the second is the ordinary pinch 
forceps with which the second cover-glass is handled in making smears. 

(i) Examination of Fresh Blood. 

The examination of fresh blood is a very important part of hematological 
work and should be a routine procedure in every case possible. If the blood 
cannot be examined for several hours after being taken, it is wise not to attempt 
the study of a fresh specimen, as so many changes will occur in such slides that 
no certain findings obtain. The information obtainable from such examination 
of fresh blood often supplements that which one may derive from a study of the 
stained specimens, as some peculiarities, such as the ameboid movement of the 
leucocytes or the motility of the malarial parasite, may be studied only in this 
way. 

Technic. 

Assuming that the slides and cover-glasses are clean and dry, the ear is 
punctured as previously described. Wipe away the first few drops of blood 
and touch the center of a cover-glass, held with the pinch forceps, to the top 
of the next drop, which should be about the size of a small black-headed pin. 
If this drop be too large the layer of blood will be too thick to permit of proper 
examination. Care should be taken that the cover-glass does not touch the 
skin. Drop this cover onto a slide, which has been warmed by rubbing or 
by passing through a flame. If the glassware be clean, the drop will spread 
evenly in a thin circular layer, not quite to the edge of the cover-glass. Under 
no circumstances should pressure be used to thin the layer or to readjust 
the cover after it has settled on the slide, as artefacts may be easily introduced 
in this way. The slides thus prepared are examined first with a low-power 
lens to obtain an idea of the even distribution of the cells over the entire area. 
The detailed study is carried out with the 3^12 oil immersion lens, but it 
should be remembered that a smaller magnification may give a better general 
survey. These preparations will keep long enough for the purposes of exami- 
nation, but if one wishes to preserve the blood fresh and uncoagulated for a 
longer period it is well to enclose the cover-glass with vaselin or paraffin or to 
use the ordinary hanging-drop chamber as suggested by Rosin and Bibergeil. 
It is sometimes desirable, especially in the study of malarial parasites, to use 
a warm stage or a warm chamber. If, however, the specimen is examined 
soon after its preparation, no such precaution is necessary, provided the 
room is not cold. 



526 DIAGNOSTIC METHODS 

In order to judge of the changes which abnormal blood may show in the 
fresh state, one must be thoroughly familiar with the appearance of normal 
blood. This latter knowledge can by obtained only by frequent study of 
fresh normal specimens and not from any text-book description. To at- 
tempt to learn without microscopic study the size, shape, color, and refraction 
of the red and white cells, the relation of the blood-plates to fibrin formation, 
the number of the various cells and their ratio to one another, would be 
absolute idiocy. 

An examination of the fresh blood as described above gives information 
regarding the presence or absence of the malarial parasite, the spirochete 
of relapsing fever, the filaria, and trypanosomes. It affords evidence of in- 
creased or decreased rouleaux formation, number, deformities, and degenera- 
tions, as well as of the amount of hemoglobin of the red cells; the presence of a 
leucocytosis or of a leucopenia and of ameboid movement of the leucocytes. 
However, care must be taken to avoid premature conclusions from such study 
and to institute further examinations of the stained specimen to clear up 
doubtful points. The observer must be on his guard lest he mistake the nor- 
mal Brownian movement in the protoplasm of the cells for ameboid or parasitic 
movement. Curious phenomena are observed in the fresh specimen as^the 
blood dries and should not be misinterpreted. The various characteristics 
of fresh blood will be taken up in detail later. The introduction of the ultra- 
condenser or dark-field illuminator has opened up a field of great possibilities 
in the examination of specimens of fresh blood, especially when malarial 
parasites or spirochaetae pallidae are suspected. 






Fig. 137. — Preparation of smears, with two-^lass slides. {Da Costa.) 

(2) Preparation of Smears. 

To prepare blood smears, which are to be later examined in the stained 
condition, one may spread the blood in capillary layers on slides or between 
cover-glasses. The former method is the one used in the writer's laboratory 
and has given excellent and satisfactory results. A fair-sized drop of blood 
is collected on one end of a clean dry slide, held between the thumb and second 
and third finger of the left hand. A second slide is held in the same manner 
by the right hand, but at an angle of 45 degrees to the first one and touching 
the drop of blood. Allow the blood to spread out by capillarity along the 
edge of the second slide. As soon as this occurs, draw the drop of blood along 
the first sUde with a clean sweep, exerting little pressure with the second slide 
and maintaining the angle of 45° between the two slides, allowing the second 
slide to rest rather upon the blood than upon the slide (see cut). In the proc- 
ess, as recommended by some writers, the second slide is gradually drawn 



THE BLOOD 527 

into a position perpendicular to the first one. This procedure does not yield, 
in the writer's hands, as good results as the former method, as it is more 
difficult to maintain equal pressure, the smear being as a result too thick or 
too thin in places. Instead of a slide, a cigarette paper may be used as a 
spreader and gives good results. This method of making blood smears has 
the advantage of offering a large surface for examination, of making smears 
which are fairly uniform after some practice, of dispensing with the necessity 
of mounting the specimen, and of permitting the fixation of the smear in the 




Fig. 138. — Preparation of blood smear with cigarette paper. (Da Costa.) 

free flame. It is less expensive than the method to be described later and 
permits of the cleaning and later use of the slides. The beginner may find, 
on examining his early specimens made by this method, that the leucocytes 
collect at the distal end of the smear and that the general surface contains 
few white cells. This is due to the use of undue pressure in making the smear, 
and may be avoided by proper attention to this detail. 

A second method which has many advocates is the use of two cover- 
glasses. One clean, dry cover-glass, which should not be too large (prefer- 
ably three-fourths inch square or the larger rectangular slips) , is held by the 
Ehrlich or cross-bladed forceps or, as some advise, between the thumb and 
first finger of the left hand. The other cover, held in the pinch forceps or 
between the thumb and first finger of the right hand, is touched to the drop^of 




Fig. 139. — Ehrlich forceps. 

blood as it wells from the puncture in the ear. This second cover is then 
dropped at once upon the first in such a way that the corners of the two glasses 
do not coincide. If the glasses are clean the blood spreads out evenly in a 
thin capillary layer between them. As soon as the spreading is complete, the 
two covers are drawn apart, in a line parallel to the plane of their surfaces, by 
a steady, quick motion, being sure to avoid lifting them apart. This manipu- 
lation can be learned only by practice and never from any description. If 
the fingers are dry and if care be taken to touch only the corners of the covers 
the forceps need not be used in separating the covers, but it must be remem- 
bered that moisture will cause changes in the specimen. It is, therefore, 
advisable to use the forceps in this part of the technic unless the rectangular 
slips be used. As soon as the covers are separated, they are allowed to dry in 
the air or by waving them two or three times to and fro. They should be at 



528 DIAGNOSTIC METHODS 

once placed in a clean, closed receptacle, such as a Petri dish, until ready for 
the later complete fixation and staining, as dust will collect upon them or 
flies may attack them if left in the open air. It is rarely necessary to fix these 
smears at once, but with some stains such treatment is advisable. This 
second method of making the blood smears is more difficult than the first, is 
not so reliable, does not give as great a surface for examination, and always 
shows the lower cover-glass better and more uniformly spread than the upper. 
Smears made by either of these methods should be uniform throughout 
with the exception of the edges, which should never be used as they are too 
thick for allowing definite conclusions to be drawn. The red cells should 
lie on their broad surface, should not be in rouleaux except at the edges, and 
should not show deformities due to errors in technic. The leucocytes fre- 
quently collect at the edges of the specimen if too great pressure be used in 



Fig. 140. — Pinch forceps. 

making the smear, while the platelets always collect at the point first touched 
by the second slide or cover-glass. The preparation of thin even smears is 
necessary for the proper carrying out of the later technic. Those specimens 
which are irregular or are too thick would better be discarded, as the time 
consumed in studying such specimens will not be compensated for by the 
results obtained. It is much better to make several smears than to be content 
with a few bad ones. A little experience with poor smears will convince the 
worker that it is advisable to use great care in preparing them, especially if 
a differential count is to be made or if a study of the degenerations and 
deformities of the cells is to be undertaken.^ 

(3) Fixation of Smears. 

Before any staining of the cellular elements of the smear takes place, the 
protein constituents of the blood must be coagulated by exposing the air- 
dried film to the action of a high degree of heat or to that of various chemical 
reagents. The selection of the method of fixation will depend to a great ex- 
tent upon the stain to be used later. Fixation is always essential if aqueous 
stains are used, while it is not so necessary if strong alcoholic solutions are 
employed. In the use of the different modifications of the Romanowsky 
stain, the fixation is done by the methyl alcohol employed as a solvent for the 
various stains. As a general rule, fixation by heat is preferable to that by 
chemicals, as artefacts are less prone to appear, providing the degree of heat 
is carefully regulated. 
Fixation by Heat. 

This method, which is the most difficult to use and which is at the same 
time the best, is the only one which is reliable when Ehrlich's triple stain is to 

1 See Schilling, Deutsch. med. Wchnschr., 1913, XXXIX, 1985. 



THE BLOOD 



529 



be employed. The principle is as follows: The air-dried specimen is sub- 
jected to the action of a temperature of 110° to 150°, for. a more or less 
varying length of time, depending on the experience of the special worker. 
The lower the temperature the longer must' its action be exerted. 

The apparatus' most frequently ejnployed is the copper plate introduced 
by Ehrlich. This is an unpolished triangular plate of copper about 3 mm. 
thick, 30 to 50 cm. long, and 10 cm. wide, which is held in position by vertical 
standards. It is heated by an alcohol lamp or a Bunsen burner placed under 
the narrower end until the temperature of the plate becomes constant, the 
parts nearer the flame being naturally warmer than those more remote. The 
temperature of the different portions of the plate may be readily found by 
determining the points at which water (100), toluol (no), xylol (140), or of 
turpentine (150) boil. The slides or cover-glasses are then placed, smeared 
side up, at the desired point (the outer margin of the glass being three-fourths 
inch from the boiling-point and toward the flame) . Just how long a period is 
necessary, at the temperature selected, to give a perfect fixation will depend 
upon the age of the specimen and upon the 
condition to be studied. It is good practice 
to place several slides at the point desired for 
a period of one hour and then remove a slide 
at intervals of 15 minutes thereafter. One 
of the specimens is sure to be good by this 
method and the remaining ones may, there- 
fore, be properly heated. Freshly made 
specimens require longer heating than the 
old ones, while normal blood requires a 
longer exposure than abnormal specimens. 
As a rule, the specimens require, when the 
triple stain is to be used (and this is the one 
most frequently employed with heat fixation) , 
from one to one and one-half hours at a tem- 
perature of no, although some workers use pj(. 
only a few minutes' (one to three) exposure to 
such temperatures. The higher the tempera- 
ture the less time is essential. Rubinstein uses a point at which a drop of 
water does not boil, but assumes the spheroidal state (so-called Leidenfrost 
phenomenon) and places the slides, with the smeared side down, upon the 
plate at this point for one-half to three-fourths minute. Some workers, as 
Pappenheim, use this point, but place the smeared side upward. 

Instead of the copper plate, one may use the ordinary drying oven or a 
Victor Meyer heater. It is the writer's custom to use a copper drying oven 
heated by a gas flame regulated by a thermostat. The slides, with the 
smeared side downward, are placed on a glass plate whose temperature is 
measured by an accurate thermometer. The temperature is allowed to in- 
crease gradually to about 80°, from which point it is more quickly raised until 
the desired stage is approximated, when the heating must continue slowly, the 





141. — Oven for fixing blood 
films. {Da Costa.) 



530 DIAGNOSTIC METHODS 

final temperature being maintained for 15 minutes. The best temperature 
for fixation, in the writer's experience, is 110° to 120° for 15 minutes when the 
staining is to be done with eosin-methylene blue or eosin-hemotoxylin, while 
for the tri-acid stain a temperature of 120° to 125° for one and one-half hours 
should be maintained. Some workers heat to 160° rapidly and then allow 
the films to cool to 30°, when fixation is complete in about 15 minutes. Engel 
and Cabot recommend, in the absence of other equipment, the passing of 
the smear through the flame several times. Such treatment often yields 
excellent results, but is uncertain and requires much experience. It is to be 
remembered that too rapid changes of temperature are to be avoided, as 
shrinking or splitting of the cells will occur under such conditions. So much 
depends upon proper fixation that a little more time spent in obtaining good 
specimens will shorten the time necessary for the future examination. Proper 
staining depends on proper fixation, especially with the tri-acid stain, in which 
cases the over- or under-heating is evident in the color tone of the erythrocytes. 

Chemical Fixation. 
(i) Absolute Alcohol. 

Allow the alcohol to act for five minutes to one hour, or boiling alcohol 
for one minute. The slide is simply covered with the fixative and left for the 
desired period. Much depends upon the stain to be used as to the time neces- 
sary for good fixation. If an alcoholic stain, five minutes is sufficient; if a 
watery or alkaline one, a longer time is essential. After fixation drain off the 
alcohol and allow the residue to evaporate in the air or wash with water and 
dry between sheets of filter-paper. If alcohoUc stains are to be used the moist 
specimen may be directly passed through the flame. This fixative is unre- 
liable if a study of the neutrophile granules is to be made, but it brings out 
the nuclear structures fairly well. 

(2) Nikiforoff's Method. 

Cover the smear with equal parts of absolute alcohol and ether and allow 
the fixative to act from one-half to two hours. After the fixation allow the 
fixative to evaporate or wash with water and dry. Some workers advocate 
short fixation especially where the malarial organism is to be studied. This 
fixative brings out the degenerations of the red cells in good shape. 

(3) Methyl Alcohol. 

This fixative is used absolutely pure for three to five minutes. It is the 
most generally applicable chemical fixative and gives beautiful specimens. 
If used in combination with stains, as in the Romanowsky methods, it gives 
as perfect preparations as absolute alcohol in one-half hour and brings out the 
neutrophile granulations in better outhne. A longer fixation than three to 
five minutes does no harm; and a shorter one, especially if the fixative is 
the solvent for the stain, will give fair results, the outline of the cells being 
sharper the longer the fixative is allowed to act. 

(4) Formalin. 

This fixative may be used as a i per cent, solution in 95 per cent, alcohol 
and allowed to act for one minute, as Benario advocates, but the writer has 



THE BLOOD 53 1 

had much better success with the 0.25 per cent, solution in 95 per cent, alco- 
hol for one minute, as Futcher and Lazear suggest. Allow the fixative to act 
for one minute, wash in water, and dry between filter-paper. Some writers 
advocate the direct treatment of the fixed smear with the stain without an 
intermediate washing and drying, but I have never found the results as good 
by this method. Instead of the formalin solutions, the vapors may be used 
by placing the specimen under a bell jar with a few drops of 40 per cent, for- 
malin and allowing the fixative to act for one to five minutes.^ The normal 
staining of the cells is not always as clear as could be desired after the use of 
such a fixative. 

A large number of inorganic fixatives have been advised, among them 
being mercuric chlorid, chromic acid and osmic acid, but these fixatives 
are much more apt to produce artefacts than are the others mentioned above. 
While these latter fixatives give good demonstrations of the nuclear structures 
and of mitotic figures, the granules are very imperfect; but chromic acid 
has many advantages as a fixative in the study of the chromatin elements. 
(4) Staining Methods. 

(A) General Considerations. 

Since the work of Witt, we have recognized that the color of an organic 
substance is due to the presence of two definite atomic complexes in the 
molecule of the colored substance. The first of these, the chromophore group, 
is responsible for the chromogenic properties of the substance, while its in- 
fluence as a dye is increased by the presence of the second or auxochromic 
group. The color of the compound is the more intense the more of these 
groups are present. Groups or atoms which intensify the color of the sub- 
stance are called, by Schiitze, bathochromes, while those which reduce the 
color are called hypsochromes. 

While the dyes, the so-called anilin dyes, used in hematological work 
are all aromatic derivatives, it must not be assumed that such are alone 
characterized by staining qualities, as many simple aliphatic derivatives show 
a definite color and exert some staining property, depending on the presence 
of the two groups above mentioned. The chief chromophore groups are the 
CO (carbonyl) group, the CS group, CN, HCN, the — N = N — (azo group), 

the yO (azoxy group), NO (nitroso group), — N^' | (nitro group), 

— N-^ ^O 

and the — N = SO group. The most important auxochrome groups are the 
NH2 and the OH groups, the former being a much more powerful one than 
the latter. These auxochrome or haptophore groups make possible the 
union of the stain with the tissue as a direct salt formation. Substances, 
which possess a chromophore group and are colored or intensified by the 
presence of the auxochrome group, are called chromogens. It is, therefore, 
evident that the effect of an auxochrome depends not only upon its own na- 
ture, but also upon that of the chromogen. Through the introduction of H 
by means of hydrocarbon radicals, new auxochromes are produced and the 
^ See van Herwerden, Nederl. Tijdschr. v. Geneesk, 1919, II, 170. 



532 DIAGNOSTIC METHODS 

color becomes intensified, the effect being so much greater the higher the 
molecular weight of the substituting hydrocarbon radical. 

As a large number of colored substances have a tendency to form tauto- 
meric compounds, the salt formed by combination with the auxochrome 
groups may have a different constitution from that of the free base or acid. 
Many chromophore groups are also capable of forming salts, but only with 
strong acids or bases. In this formation of salts, by union with the auxo- 
chrome or chromophore groups, we must remember that halochromia may be 
seen. By this is meant that uncolored or weakly colored substances may 
combine with acids to form salts without the color being due to the presence of 
a chromophore group. It is essential that the chromophore group possess a 
double bond of union, as oscillation in one portion of the molecule is thus 
possible. As the chromophoric as well as the auxochromic radicals may have 
acid or basic tendencies, it is manifest that the reaction of the substitution 
product will depend upon the interrelations of the acid and the basic radicals. 
The best dyes are, therefore, obtained by substitution in one direction, that 
is, by accumulating either basic or acid groups separately. 

It is thus seen that we have two general classes of dyes, the acid and 
basic, depending upon the preponderance of the total acid or basic groups in 
the molecule of the dye. It has been shown by Ehrlich that these acid and 
basic dyes, may be so combined that a third one results, showing neither acid 
nor basic properties. This class of dyes, known as neutral dyes, is of the 
greatest importance in hematological work. In them we have not only the 
staining properties due to the original chromophore and auxochrome groups 
of both the acid and basic dyes, but also those due to the union of the compo- 
nent groups in the neutral dye. Such dyes are hence called polychrome dyes 
and are usually soluble in an excess of one of the component mother dyes, 
generally the acid one. It must be understood that in speaking of a dye as 
acid, basic, or neutral, we do not refer so much in our staining work to the 
chemical reaction of the dye, but to the portion of the dye to which the stain- 
ing is due — that is, to the specific auxochrome and chromophore groups. 

Among the basic stains we find methyl green, methylene blue, amethyst 
violet, neutral red, dahlia, pyronin, thionin, fuchsin, methyl violet, Bismarck 
brown, alum hematoxylin, and safranin. Most of these stains, depending 
on the strength of their component groups, color the nuclear substance of both 
red and white cells, as also the cytoplasm and certain granules of abnormal 
red and of some normal white cells, the staining being influenced, as Matthews 
has shown, by the alkaline reaction of the tissues. The most important acid 
stains are eosin, acid fuchsin, orange-G, indulin, nigrosin, aurantia, and salts 
of picric acid. These dyes color the red cells and the eosinophile (oxyphile) 
granulations of the leucocytes. The neutral portion of the dyes color the so- 
called neutrophile granules of the leucocytes. 

In the process of staining, it is a question whether we have to do with 
purely chemical processes as Knecht's theory assumes or whether the solid- 
solution theory of Witt or the mechanical theory of surface attraction are 
accountable for the phenomena observed. It is probable that the salts of the 



THE BLOOD 533 

dyes are decomposed by the cells and that hew compounds result from the 
union of the acid and basic stains and the various chemical entities of the 
cells. Yet we have instances in which the dye is simply stored in the cell 
without any chemical union taking place. We must, however, account for 
the elective character of certain stains by a purely chemical activity, as, for 
instance, chromatin, which undoubtedly consists of nucleinic acid, always 
takes a basic stain, even though a neutral compound is used as the staining 
agent. It is to be remarked that a neutral stain does not color all of the acido- 
phile or basophile substances of the cells of the same tint. Thus, eosinophile 
granules are differently colored from the oxyphile hemoglobin of the red cells. 
The neutral portion of the stain has nothing to do with the staining process 
beyond the coloring of the neutrophile substances in the protoplasm of certain 
leucocytes. 

In the selection of a stain for general hematological work, it is necessary 
to use a compound stain or, at least, two simple stains, one after the other. 
A single simple stain colors only a few of the elements and affords no general 
idea of the blood picture. Indeed, to obtain definite conceptions of the finer 
structure of the blood, it is necessary to study several slides stained by differ- 
ent methods. It is usual to select for routine work a stain which will reveal 
the greatest amount of information regarding the histological characteristics 
of the blood. This is the so-called panoptic staining and is to-day generally 
accomplished by the use of the various forms of neutral stains. In making 
the stains for one's self or in buying them in the market, one must be certain 
that only chemically pure pigments are used and that the solution is made 
according to the f-ormula with the purest solvent obtainable. It will need 
only one experience with a poorly made stain to convince the worker of the 
importance of this detail. 

(B) Methods of Staining. 

In his work the author uses, for routine purposes, the Wright's stain, 
the eosin-methylene blue, and eosin-hematoxyhn stains. These stains have 
the advantage of giving clear-cut pictures of practically all of the important 
blood elements along with simplicity of technic. As it is not always pos- 
sible to secure or even to make a Wright or an Ehrlich triple stain which will 
give reliable results under all circumstances, the writer feels that the general 
worker would better use the eosin-methylene blue stain for his daily work. 
The many modifications of the Romanowsky stain have their advocates, and 
it must be said that they yield reliable results when they are working properly, 
but no one can say when they will go wrong or how to make them work right 
when once they do give poor results. 

Eosin-Methylene Blue. 

A number of methods of using these two simple stains have been advo- 
cated by such workers as Chenzinsky, Ehrlich and Lazarus, von Willebrand, 
Plehn, Aldehoff, and Gabritschewsky, but the writer has found the method 
advocated by Miillern^ to be the most generally applicable and if properly, 

^ Grundriss der klinischen Blutuntersuchung. Wien, 1909. 



534 DIAGNOSTIC METHODS 

applied, the most reliable modification. By it we are able to stain all of the 
blood elements, including the neutrophile granules and obtain, thus, a 
panoptic picture whose findings are not excelled by those of the various modi- 
fications of the Romanowsky stain. 

Technic. 

(a) Fixation of the smear in pure methyl alcohol for three minutes, (b) 
Preliminary staining in J^ per cent, alcoholic (70 per cent.) solution of 
Griibler's ^'french pure" eosin from three to five minutes, (c) Wash in dis- 
tilled water and dry between filter-paper, (d) Lay the slide in a carefully 
measured and well-mixed solution of 20 drops of J-^ per cent, aqueous solution 
of methylene blue (B. pat.) and ten drops of the above eosin solution for one- 
half to one minute, (e) Wash quickly and briefly with distilled water and 
dry at once between filter-paper or over the flame. (/") Mount if desired in 
Canada balsam or examine directly with a high-power lens. 

This stain shows the red cells and the eosinophile granules of the leuco- 
cytes of a bright red tone, the neutrophile granules pink to bright red (distin- 
guished from the eosinophiles by their smaller size), the nuclei, mast-cell 
granules, bodies of the lymphocytes, platelets, malarial organisms, tr5^ano- 
somes, and filaria varying shades of blue. The preliminary staining with 
eosin serves the purpose of bringing out the neutrophile granules more clearly 
while the basophile granulations are of course unaffected by such treatment. 
It is probable that the later staining with the eosin-methylene blue mixture 
has the same characteristics as the neutral stains to be mentioned later. While 
this stain has the advantage of simplicity, reliability, and panoptic power of 
staining, it is somewhat inferior to other stains in bringing out some forms of 
the malarial organism, owing to its lack of chromatin staining qualities. The 
writer has found that the eosin and methylene blue above mentioned are the 
best to use in this process and that Turk's advice, regarding the use of fairly 
fresh eosin solutions and of old methylene-blue solutions, is warranted. The 
blood preparations should not be over a few days old to show the best pictures, 
as those over a week old may show a diffuse plasma staining and the neutro- 
phile granules not clearly differentiated. If the nuclei do not stain well with 
the methylene blue as many writers claim, make a second preparation fixing 
somewhat longer in methyl alcohol. By following out these precautions the 
worker will be rewarded with beautiful specimens. This method is to be 
recommended for all routine examinations. 

Eosin Hematoxylin. 

This stain is especially important in cases in which the nuclear structures 

are to be studied.^ It stains the nuclei beautifully, showing their finer 

structure, karyokinetic figures, and pycnotic qualities, as well as the basophile 

granules of both red and white cells. The solutions required are: (i) H P^r 

cent. Griibler 's blood eosin in 70 per cent, alcohol. We may use, with equally 

good results, the eosin mentioned in the previous method. (2) Delafield's 

hematoxylin, the formula of which is 

^ See Williams (Jour. Am. Med. Assn., 1913, LXI, 1627), who describes a modificationi 
of this stain which he calls "invariable." 



THE BLOOD 535 

Hematoxylin crystals, 4 grams. 

Alcohol (absolute), 25 c.c. 

Ammonium-alum crystals, C. P., 52 grams. 
Distilled water, 400 c.c. 

Glycerin, C. P., 100 c.c. 

Methyl alcohol, C. P., 100 c.c. 

Rub up the hematoxylin crystals with the alcohol until they are dissolved 
and place the solution in a loosely corked glass bottle, allowing it to stand 
exposed to the hght for four days. Dissolve the ammonium-alum in the 
water and allow it to stand exposed in the same way for four days. At the 
end of this time mix the two solutions, shake thoroughly, and filter at the end 
of three hours. Add the glycerin and methyl alcohol to the filtrate and allow 
this to stand overnight. Filter the mixture, place it in a clear bottle, and 
allow it to ripen, exposed to the light for six weeks, when it is ready for use. 

Technic. 

Stain the specimen with the eosin solution for one-half minute, and wash 
in water. Without drying place the slide in the hematoxylin solution for 
one to three minutes, the time varying with the particular stain and with the 
experience of the worker. Wash with water, dry, and mount. This stain 
does not give as good results as does the former method, but is to be espe- 
cially recommended when the nuclear structures are to be studied. 

Ehrlich's Triple Stain. 

In the literature of hematology we find the expressions "triacid" and 
"triple stain" used synonymously. The triacid stain, as originated by 
Ehrlich, was a mixture of equal parts of saturated solutions of indulin,, 
nigrosin, and aurantia, and was used to differentiate the eosinophile granules. 
When used synonymously in these days, the triple stain is always meant. 
The composition of this latter stain is as follows, made up of two acid and 
one basic stain: 

Saturated watery solution of orange-G, 13-14 c.c. 

Saturated watery solution of acid fuschin, 6-7 c.c. 

Distilled water, 15 c.c. 

Alcohol (absolute), 15 c.c. 
Saturated watery solution of methyl green 00, 12.5 c.c. 

Alcohol (absolute), 10 c.c. 

Glycerin, 10 c.c. 

The pigments must be chemically pure and the solutions must be added 
in the order named, the methyl-green solution being added drop by drop with 
constant stirring. 

This stain is difficultly prepared, being usually a failure. The writer 
would, therefore, advise the worker to obtain it already made; and even then 
it may not prove satisfactory. The correct stain should have a russet-brown 
color and should not show any trace of a precipitate. It improves up to a 



536 DIAGNOSTIC METHODS 

certain point with age but, after a time, precipitates invariably occur, making 
it useless. It should never be filtered or shaken, the solution to be used being 
taken from the center of the bottle with a dropping pipet. ) 

Technic. 

The smear should be fixed by heat as previously described. After proper 
filiation, cover the smear with the stain and allow it to act for one to ten min- 
utes as the experience of the worker with the particular stain may indicate. 
Wash with distilled water, dry, and mount. One advantage of this stain is 
that it cannot overstain, those films appearing so being underheated, while 
those understained are overheated. 

The stain shows the red cells of a buff or orange color, without the 
slightest shade of red (a reddish tint is given with underfixed specimens while 
a yellow tone is shown by those overheated), the nuclei of the leucocytes 
a dark green, those of the normoblasts black, the neutrophile granules a lilac 
color (though some occasionally show a reddish tinge), and the eosinophile 
granules of a crimson tone. 

This stain was introduced as specific for the neutrophile granules, but its 
disadvantages are too numerous to warrant its recommendation as a routine 
stain. It is a poor nuclear stain, does not show the structure of the normal 
mononuclear leucocytes, does not stain the basophile granules, nor the malarial 
or other parasites. For a reliable preparation, showing those features for 
which it is especially adapted, a proper fixation is an absolute essential. 

On account of the lack of nuclear staining with the triple stain, Pappen- 
heim has substituted methylene blue or methylene azure for the methyl green, 
and eosin for the acid fuchsin. The writer has had no experience with this 
modification and cannot, therefore, speak regarding its value. 

Polychrome Methylene -blue -eosin Stains. 

These stains are very numerous, each having its advocates. They are 
easy to use, contain a rehable fixative, and give satisfactory results, but are 
not always obtainable or easily prepared. They are the stains which give, 
perhaps, the best panoptic results and are especially serviceable in the study 
of the malarial organism and other parasites as many of them contain chroma- 
tin-staining elements. The granulations of the leucocytes are not as well 
marked in all cases as they should be, so that for a complete study of the 
various types of granules in the cells several stains would better be used. 

Romanowsky had found that the addition of a watery eosin solution to 
an aqueous methylene-blue solution, until an insoluble precipitate began to 
form, gave rise to new staining properties of the solutions, in the sense that 
the chromatin substance of malarial organisms was stained a beautiful red. 
The specific staining properties of this mixture were later found to be due 
not to a combination of eosin and methylene blue, per se, but rather to the 
formation of a new compound between eosin and an impurity or decomposi- 
tion product in the methylene blue, namely, methylene azure. Jenner in his 
stain makes use of a methyl-alcohol solution of the isolated precipitate, the 



THE BLOOD 537 

eosinate of methylene blue, which lacks the red chromatin staining element. 
The same may be said of the May-Griinwald stain. 

In making up these polychrome stains it is not general to use the pure 
methylene azure and eosin, but rather solutions of methylene blue containing 
a variable amount of the methylene azure to which eosin is added. Recently 
Wilson/ in a careful study of the methylene-blue-eosin combinations, has 
shown that very little methylene azure and methylene violet exist in the stains 
as commonly employed. He finds evidences of at least four staining bodies in 
such mixtures, namely, the eosinate of methylene blue, eosinate of methylene 
violet, eosinate of methylene azure, and eosinate of thionin. 

Wright's Stain. 

This stain is a much improved modification of the Leishman stain, ^ as its 
method of preparation requires but a few hours and as the resulting dye is 
somewhat more panoptic than is that made by the Leishman method. 

Preparation. 

To a 0.5 per cent, aqueous solution of sodium bicarbonate add methylene 
blue (B. X. or ''medicinally pure") in the proportion of i gm. of the dye to 
each 100 c.c. of the solution. Heat the mixture in a steam sterilizer at ioo°C. 
for one full hour, counting the time after the sterilizer has become thoroughly 
heated. The mixture is to be contained in a flask, or flasks, of such a size and 
shape that it forms a layer not more than 6 cm. deep. After heating, allow 
the mixture to cool, placing the flask in cold water if desired, and then filter 
it to remove the precipitate which has formed in it. It should, when cold, 
have a deep purple-red color when viewed in a thin layer by transmitted 
yellowish artificial light. It does not show this color while it is warm. To 
each 100 c.c. of the filtered mixture add 500 c.c. of a o.i per cent, aqueous 
solution of "yellowish, water-soluble" eosin and mix thoroughly. Collect 
the abundant precipitate, which immediately appears, on a filter. When the 
precipitate is dry, dissolve it in methyl alcohol (Merck's "reagent") in the 
proportion of o.i gm. to 60 c.c. of the alcohol. In order to facilitate solution, 
the precipitate is to be rubbed up with the alcohol in a porcelain dish or mor- 
tar with a spatula or pestle. This alcoholic solution of the precipitate is the 
staining fluid. It should be kept in a well-stoppered bottle because of the 
volatility of the alcohol.^ 

Technic. 

Cover the film with a noted quantity of the staining fluid by means of a 
medicine dropper. After one minute add to the staining fluid on the film the 

^ Jour. Exper. Med., 1907, IX, 645. 

2 Leishfnan Stain. Prepare a i per cent, aqueous solution of medicinal methylene blue 
(Griibler) and add 0.5 per cent, sodium carbonate; heat for 12 hours at 65°C. and keep for 
10 days at room temperature. Prepare a 0.1 per cent, aqueous solution of "yellowish, 
water-soluble" eosin. Mix equal volumes of the two solutions and leave for 10 to 20 hours, 
shaking the mixture at frequent intervals. Filter, wash the precipitate with distilled water 
until the washings are a pale blue, dry on the filter and powder the dried residue. The 
staining solution is prepared by dissolving 0.2 gram of this powder in 10 c.c. of methyl 
alcohol. This stain is used precisely as the Wright stain, the blood-picture being very 
similar. 

^ Jour. Am. Med. iVssn., 1910, LV, 1979. 



538 DIAGNOSTIC METHODS 

same quantity of distilled water by means of the medicine dropper and allow 
the mixture to remain for two or three minutes, according to the intensity of 
the staining desired. A longer period of staining may produce a precipitate. 
Eosinophilic granules are best brought out by a short period of staining. The 
quantity of the diluted fluid on the preparation should not be so large that 
some of it runs off. Wash the preparation in water for thirty seconds or until 
the thinner portions of the film become yellow or pink in color. The experi- 
ence of the worker may be such as to demand longer decolorization with any 
particular specimen. When the desired degree of differentiation is reached, 
dry the specimen quickly between filter-paper, mount if desired, and examine 
first with the low-power lens to observe the staining effects, and then with the 
high-power lens for the more minute study. When searching for malarial 
parasites, the decolorization would better be of short duration, as the chroma- 
tin suffers to a great extent in this process. 

This stain shows the red cells colored either orange or pink (depending 
on the time of decolorization), nuclei of leucocytes blue or dark lilac, neutro- 
phile granules lilac, eosinophile granules red or pink, fine basophile granules 
deep blue, large mast-cell granules purple, protoplasm of the lymphocytes 
robin 's-egg blue, blood-plates deep blue or purple, bacteria blue, malarial 
and other parasites blue, the chromatin element varying from lilac to ruby- 
red to black. Polychromatophilia and granular degenerations are well 
shown, the granules being blue. This stain is very useful in studying lympho- 
cytes, mast cells, blood-plaques, and the finer structure of the malarial organ- 
ism; but the leucocytic granules, at times, are not sufficiently differentiated. 

Giemsa Stain. 

Giemsa has shown that the complicated methods of preparing the dyes 
may be dispensed with by the use of the pure staining substance extracted 
from the polychrome methylene blue, namely, the methylene azure. If this 
pure pigment be used the stain becomes a pure chromatin one. It is advis- 
able, therefore, to use Griibler's azure II, a combination of equal parts of 
methylene azure and medicinal methylene blue. The formula of the Giemsa- 
stain is as follows: 

Azur II-Eosin, 3.0 grams. 

Azur II, 0.8 gram. 

Glycerin (C. P.), 125.0 grams. 

Methyl alcohol (C. P.), 375.0 grams. 

Grind up the dyes in the alcohol and then add the glycerin. 

Technic. 

Fix the films in methyl alcohol, and stain for fifteen minutes in a mixture 
of 15 drops of the stain to 10 c.c. of distilled water. If necessary, a trace of 
sodium carbonate may be added to the water to intensify the basic stains. 
Wash in water, dry, and mount. It is to be remembered that oil of cedar 
will bleach these specimens quite rapidly , so that it is advisable, if the films are 
to be kept, to stain them upon the slides so that mounting becomes unneces- 
sary. Strong light should not be allowed to act on these stained films for any 



THE BLOOD 539 

length of time as the chromogen stain fades rather quickly under these condi- 
tions. The various elements are stained as with the Wright stain, but the 
neutrophile granules are often not well defined J 

Pappenheim's Stain. 

Pappenheim (Technik der klin. Blutuntersuchungen, Berlin, 191 1; Folia 
Hsemat., 191 2, XIII, 339) advocates as the best and most serviceable stain 
the combination of the Giemsa stain as follows: Fix the blood film by covering 
it with May-Griinwald stain (Wright's answers as well) for 3 minutes, add to 
this stain on the slide an equal amount of distilled water and allow it to act for 
I mini. ce. Pour off the fluid but do not wash. Now cover the stained film 
with dilute Giemsa solution (15 drops of stain to 10 c.c. of distilled water) and 
allow to act for 1 5 minutes. Wash thoroughly and dry, but not over the flame. 
Mount, if desired, in neutral Canada balsam. The result is extremely pan- 
optic. The nuclei stain violet. The lymphocytes, monocytes and undiffer- 
entiated lymphoidocytes have a delicate blue cytoplasm. The mast-cell 
granulations are ultramarine blue. Eosinophiles have brick-red granulations 
and the neutrophiles a rose-colored cytoplasm, while their granules are 
indefinite. 

Specific Stains for Malarial Organisms. 

While the above stains all give good results with the malarial organisms, 
enabling one to make a diagnosis from an examination of the smear, yet 
certain other stains are often of advantage in that little else is stained and 
thus the confusion arising from indefinite staining is obviated. 

Thionin Stain (Futcher and Lazear). 

Add to 100 c.c. of 2 per cent, carbolic acid 20 c.c. of a saturated solution 
of thionin (Lauth's violet) in 50 per cent, alcohol, and allow this mixture to 
ripen for a few days.^ Fix the specimen by the formalin method given above 
and stain the smear for 10 to 15 seconds. Wash in water, dry, and mount. 
These preparations do not keep indefinitely, usually fading within a year. 

The Plasmodia are shown as deep purple, irregular masses enclosed in 
the faint green red cells. The hyaline forms show as reddish-violet ring-like 
bodies. The basophile granules and the nuclei are the only other elements 
showing any particular staining qualities. 

Nocht's Stain. 

This is essentially a Romanowsky stain and is made as follows: Add 
two or three drops of i per cent, aqueous eosin solution to 2 c.c. of water. 
To this diluted eosin solution polychrome methylene blue is added, drop by 
drop, until the red color of the eosin is only faintly visible (the polychrome 

^ See Mcjunkin (Jour. A. M. A., 1915, LXV, 2164) for a new polychrome stain. Also, 
see Martiri. Rif. Med., 1916, XXXII, 1158; Krauss, Jour. Lab. and Clin. Med , 1916, II, 
138; Lago, Porto Rico Med. Assoc. Bull., 1919, XIII, 62; Agulhon and Chavannes, C. R. 
soc. biol. Paris, 1919, LXXXII, 149, Mcjunkin (Jour. A. M. A., 1920, LXXIV, 17) has 
devised a benzidin-polychrome stain which permits of great accuracy in the identification 
of the leucocytes, as the granules of the neutrophils, endothelial leucocytes and eosinophiles 
stain dark brown. 

2 See Lesieur and Jacquet, C. R. soc. biol., Paris, 1919, LXXXII, 267. 



540 DIAGNOSTIC METHODS 

methylene blue is a i per cent, solution of methylene blue chemically changed 
by heating with 3^ per cent, sodium carbonate solution for two days at 55°C.). 

Technic. 

Fix the film in methyl or ethyl alcohol and lay it face downward in the 
above mixture for five to ten minutes. Wash with water, dry and examine. 
The picture is the same given by Wright 's stain, except that the nuclear trans- 
formations and the chromatin substance are better differentiated, being stained 
a bright red color. 

The many other stains for the examination of fixed specimens will be passed 
over, as the writer finds the above stains applicable to practically all routine 
work. Many writers prefer different stains from those mentioned, but these 
stains do not seem to have any advantages which would warrant their use to 
the exclusion of those given above. Special stains used for bringing out 
certain granules and deformities will be discussed under later headings. 

Vital Staining. 

Although the examination of stained specimens of the blood is usually 
made with the dried and fixed smear, excellent results obtain when the fresh 
blood is stained without previous fixation. It is true that a '^ vital" staining 
of the blood-cells does not actually take place, as the dyes are decolorized 
by the reducing and oxidizing action of the living cells. However, a "post 
vital" staining, that is the staining of whole cells or portions of the cells, after 
their removal from the circulation and before the death of the cell results, 
may be accomplished in several ways. 

We may either add to the fresh drop of blood a few crystals of the stain, 
as advised by Arnold, and note the staining of certain leucocytic granules 
and nuclei and recticular structure of many erythrocytes; or we may first dry 
a staining solution upon the slide, cover this dry stain with a. drop of fresh 
blood, adjust the cover-glass, and seal this to the slide with wax. Some 
workers prefer to use the hanging-drop shde in the preparation of these fresh 
specimens. 

The stains which may be used for vital staining of the nuclei, granules, and 
plates are methylene blue, toluidin blue, thionin, neutral violet, Capri blue, 
Nile blue, brilliant-cresyl blue, Janus green, and paraphenyl blue. Of the 
protoplasmic stains we have fuchsin, acridin red, pyronin, safranin, and 
neutral red. 

While the results derived from this method of staining are not as numerous 
as those of the more usual method, yet it affords much valuable information 
regarding the vital properties of the cells and regarding the normal structure 
and the circulatory changes of the cells. ^ This method has been used exten- 
sively by Ito, Rosin and Bibergeil, Levaditi, Cesaris-Demel, and Pappenheim. 
Their results lead us to assume that further study will give us much valuable 
information concerning details of structure both of the cell and of the nucleus. 
It may be possible by this method to differentiate between certain forms of 

^ See Luzzatfo and Ravenna (Folia Haemat., 191 2, XIII, 102) for a discussion of the 
numerical relations of the granular and non-granular erythrocytes as shown by this method. 



THE BLOOD 



541 



degeneration of the cell which are now known only indefinitely under the 
names of metachronfatic and polychromatic staining.^ 

(5) Erythrocytes. 
(A) Appearance and Structure. 

In fresh, normal blood the red cells, or erythrocytes, appear as thin, 
flattened, homogeneous, biconcave, nonnucleated, discoid bodies with a 
sharply defined regular outline and a clear semitransparent center. In some 
cases they may appear distinctly cup- or bell-shaped. ^ The cells show, when 
examined singly, a pale greenish-yellow color but, when more thickly grouped, 
exhibit a reddish tint. The degree of color in these cells depends upon their 
hemoglobin content, the clear central area becoming larger and the entire cell 




Fig. 142. — Normal blood showing rouleaux formation and fibrin network. (Da Costa.) 



becoming paler as the hemoglobin decreases. The loss of hemoglobin may be 
so great as to lead to the formation of the so-called "pessary" form, in which 
only the periphery of the cell is apparent. This central pale area varies much 
in individual cells and is not at all evident in those which are flattened out. 
In dry specimens, if thinly spread, the cells are circular, the normal bicon- 
cavity is obliterated, and a uniform stain is observed. If the smear be thick 
the central pale area is observed. 

In chlorosis and secondary anemias, the color of the cells is usually uni- 

1 See Ross (Induced Cell-reproduction and Cancer, Philadelphia, 1911, and London, 
191 2) for the use of agar films containing stains and activating salts (auxetics) in this connec- 
tion. See, also, Anitschkow, Med. Klin,, 1914, X, 465; Barbaro, Gazz. d. osp., 1914, 
XXXV, 681; von MoUendorff, Deutsch. med. Wchnschr., 1914, XL, 1839; Hober, Biochem. 
Ztschr., ipi4,LXVII, 42o;Lindborn, Nord. Med. Arch., 1914, XL VII, i; Evans and Schule- 
mann. Science, 1914, XXXIX, 443; Folia Hsemat., 1915; XIX (I), 207; Rubino, Rif. Med., 
1915, XXXI, 533; Traube, Biochem., Ztschr., 1915, LXIX, 309. 

^ See Jordan, Proc. Soc. Exper. Biol, and Med., 1915, XII, 167; Arey, Science, 1916, 
XLIV, 392; Mas y Magro, Sig. Med., 1916, LXIII, 466 and 482. 



542 DIAGNOSTIC METHODS 

formly paler than normally, while in pernicious anemias the color may be even 
deeper than normal. These are the usual but not invariable pathologic 
findings. In malarial conditions, discolored cells are often observed in the 
fresh specimens, the bronzed or "brassy" tone often drawing attention to the 
presence of a parasite of the estivoautumnal type or of the quartan form. 

The red cells show a marked tendency to cohere to one another in more 
or less regularly arranged piles, forming long rows (rouleaux), hke. rolls of 
coin piled up face to face. The exact cause of this phenomenon is unknown, 
but it may be dependent on the presence of the fatty membrane surrounding 
the cell, as Peskind's findings show that the red cells are enveloped by a layer 
composed of lecithin, cholesterin, and a nucleoprotein. ^ In certain pathologic 
conditions this normal rouleaux formation is increased and in certain ones is 
decreased. The diminished rouleaux formation is observed in conditions 
associated with increased viscosity of the cells, as observed in most inflam- 
matory diseases and in the anemias due to malignant disease. This h)rper- 
viscosity of the cells has not at present much clinical significance, but may 
be shown to be of importance when our knowledge concerning the normal 
viscosity of the blood becomes more extensive. 

The structure of the erythrocytes is still an unsettled point in hematology. 
Neither membrane nor stroma have been fully demonstrated yet after the 
hemoglobin has been removed from the cells by hemolysis, a stroma may 
be definitely seen which could hardly be called an artefact. Schafer assumes 
that the hemoglobin is held in firm combination by chemical union with other 
albuminous constituents of the cell and supported by a stroma similar to that 
of Peskind's outer membrane. That some sort of an outer membrane does 
exist would seem to be proven by the experiments on hemolysis which have 
shown that many substances penetrate the cell producing hemolysis, while 
others in the same concentration have no such effect. It is hard to believe 
that a selective vital activity is at work here, as the results follow too closely 
the laws of physical chemistry as apphed to diffusion and osmosis. The cor- 
puscles are very elastic and contractile so that rapid and marked temporary 
distortions of shape are possible under the influence of variations in the com- 
position of the circulating plasma. 

As the blood dries various changes in the appearance of the red cells are 
observed. These changes, known as crenation, are due to the evaporation 
of water and depend upon the quantity of air which comes in contact with the 
specimen as well as upon the length of time this influence acts. " The develop- 
ment of one or more small, bright, highly refractile spots in the body of the 
cell and a slight indentation of the periphery of the cell are the most conspicu- 
ous indications of beginning crenation. As the process goes on, more and 
more of these hyaline points develop, until finally the whole surface of the 
corpuscle becomes thickly studded with glistening bead-like spines. As the 
stroma becomes drier and drier, its typical biconcavity and sharply cut out- 
line are lost, contracting strands of the stroma are seen to extend from point 
to point among the beaded projections, the periphery of the cell changes to a 

^ See Wiltshire, Jour. Path, and Bacteriol., 1913, XVII, 282. 



THE BLOOD 543 

cogged rim, and finally the cell becomes shrunken and shriveled up into a 
small, many-starred asterisk. Some of the erythrocytes become fragmented 
and small bits of their stroma are observed to break off and float through the 
plasma. Others become progressively paler and paler, as the hemoglobin is 
dissolved out, until complete decoloration occurs. Still others become dis- 
torted into designs of every conceivable shape so that their resemblance to 
the normal cell becomes more remote " (Da Costa) . These changes must not be 
confused with those occurring as a result of pathological changes. Crenation 
is often induced more rapidly than in normal blood, in the blood of persons 
suffering from acute infection and from chronic diseases . True ameboid move- 
ment of the red cells is sometimes seen as a result of a high-grade anemia.^ 

(B) Size and Shape. 

The average diameter of these normal human erythrocytes (normo- 
cytes) is 7.5 fx, the normal variations being between 6 and 9 )u (a micromilli- 
meter, Jfooo of a millimeter). The size varies depending on the method of 
preparing the specimen and also upon the osmotic tension of the plasma. 
Although dwarf and giant cells may occur to a slight degree at all ages, the 
normal infant blood shows these variations more markedly (3.3 to 10.3 /x, ac- 
cording to Hayem). According to Hamburger, the cells are shghtly larger 
in the venous than in arterial blood. Gram states that the size of these 
cells varies with climatic conditions, being greater in those of the northern 
cooler countries than in those of the southern warmer climates. The varia- 
tions in the size of the red cells is very sHght in the different sexes. 

Pathologically, variation in the size of these cells is a common and 
important feature. Generally speaking, variations in the cellular size indi- 
cate a severe and chronic anemia, while in the more mild acute forms of ane- 
mia such variations are unusual. The average size is said to be increased in 
jaundice, cholera, lead-poisoning, leukemia, congenital heart disease, and 
cretinism. 

Types of Pathological Erythrocytes. 

Variation in the normal size of the red cells is indicated by the term 
anisocytosis. This term does not include the misshappen red cells which 
appear in the blood as a result of degeneration or of mechanical injury and 
to which are given the name of poikilocytes. 

Microcytes. 

These are cells under the normal size, the variations being between i ju 
and 6 ju, the usual representatives being about 3.5 iU. It cannot be stated 
at present whether these cells are mere schistocytes (fragments of larger cells) 
or are perfect cells of degenerative origin. They occur normally in the blood 
of embryos and infants, but are rare in that of the adult except in patho- 
logical conditions. It is certainly true that these undersized cells may 
arise from purely physical causes, as a result of increased osmotic pressure of 
the plasma as well as from division of undersized mother cells. This latter 

^ See Krizenecky, Ztschr. f. allg. Physiol., 1915, XVII, i. 



544 DIAGNOSTIC METHODS 

phase is made possible by the appearance, especially in pernicious anemia, of 
nucleated reds of corresponding size. 

Pathologically, these smaller cells are observed in all severe anemias. 
As a rule, they stain deeply and evenly, but in some cases of pernicious anemia 
these cells are deficient in hemoglobin and show irregular staining, yet many 
of them may have an increased hemoglobin content. Occasionally, but not 
invariably, we find these undersized cells in chlorosis, in which the hemoglobin 
is deficient. At times these cells may show polychromatophiha, but this is 
not the rule. According to Tallqvist, an increase in the number of these 
microcytes (microcytosis) is an indication of rapid destruction of blood. 

Macrocytes. 

These are cells above the normal size, the variations being between lo 
and 20 fi. Those cells from 9 to 12 /z are called macrocytes; those between 
12 and 16 fj. are known as megalocytes; while those above 16 ju are termed 
gigantocytes. 

These cells are of regular shape, of even staining qualities, and generally 
without a well defined central clear area. The larger size of these cells may 
be partly due to the swelling incident to a lowered osmotic tension of the 
blood, but more probably this increase in size is traceable to the origin of the 
macrocytes from the large nucleated reds of the bone-marrow. These cells 
may show an excess or deficiency in hemoglobin, the former characteristic 
being observed in the primary pernicious anemias, while the latter is evident 
in the secondary forms. Morris and Thayer^ report ameboid movements in 
these cells. 

Pathologically, the presence of these various forms of macrocytes, giving 
rise to the condition of macrocytosis, indicates a severe and unusually chronic 
anemia. They are most frequently seen in pernicious anemia, in which the 
largest cells are sometimes the darkest and some of the microcytes are exceed- 
ingly pale. These large cells do occur, however, in leukemia, cholemia, and 
chlorosis, being frequently pale or ''chlorotic" and ''dropsical." These " drop- 
sical" cells are not sufficiently numerous, however, to change the volume 
index, as Capps has shown, of a secondary anemia to one shown in true 
pernicious anemia. 

Poikiloc3rtes. 

These are misshapen red cells of large or of small size, the varieties of 
such deformities being numerous. The presence of poikilocytes in the blood 
is known as poikilocytosis (first described by Damon) and is closely related 
to crenation as in both cases the cells may be similarly misshapen. The 
former is a pathologic process demonstrable the moment the blood is taken, 
while the latter is a physiological process appearing only after the blood 
has been in contact with air for some time. 

Poikilocytes arise in several ways. First, faulty technic, especially 
pressure on the cover-glass of the fresh specimen, will give rise to fragmenta- 
tion of some of the corpuscles into small spherical masses, dagger-shaped 
1 Arch. Int. Med., 191 1, VIII, 581. 



PLATE XVIII. 




b \ '^^ 






if-. 



IS. 



f t 



iS. 






^#^ 

?%■? 



21, 



^ 



22. 



^. 




2.V= 



Kdihai^ine Mill 



Types of Red Cells. 
(Wright's Staix. Zeiss Ocular 4, Oil Immersion Objective.) 

I — Normal Cells. 

2-3-4 — Normal Cells as seen with different Focus. 

5 — So-called "Pessary Form." 

6-7-8 POLYCHROMATOPHILIC CeLLS. 

9 — JSIacrocyte. 
10-12-13 — Poikilocytes. 

1 1 poikiloblast. 

14-15 — ^Punctate Basophilia in Red Cells. 

16-17 — Normoblasts. 

18 — Normoblast \^t;th Pycnotic Nucleus. 

19 — Punctated Normoblast. 

20-23 — Megaloblasts. 

24 — Gigantoblast. 



THE BLOOD 545 

bodies, and small elongated rods (pseudo bacilli of Hay em). Such fragmen- 
tation is indicative of lowered vitality and feeble powers of resistance of the 
cells. If the cover-glass be moved after the cells have spread, a large number 
of them will be distorted into oval or pear-shaped forms, the long axes of 
which usually point in the same direction. Secondly, true poikilocytes, or 
cells misshapen while in the circulation, are probably due to ameboid motion 
of a portion or the whole of a cell or to alterations in the plasma. These mis- 
shapen cells are usually pear-shaped with a budded projection at one or 
more poles. Cells may be seen which resemble a tennis-racket, a kidney, 
tomahawk-blade, dumb-bell, or anvil, while oval forms are especially observed 
in pernicious anemia and are considered by Cabot of diagnostic importance. 
Poikilocytosis is an indication of severe anemia with degenerative changes in 
the red cells; although it is not characteristic of any single disease, it is found 
more frequently in pernicious anemia and leukemia. 

(C) Nucleation. 

Nucleated red cells may be considered pathological at any period of 
extrauterine life, although they are usually found in the blood of the child 
during the first few days of life. These nucleated reds are always found in 
the bone-marrow, the normal and large forms being quite distinctive. The 
large form is the oldest and gives rise by cell division to the smaller cell or the 
normoblast. It is probably true that the nonnucleated red cells are derived 
from the nucleated form, but the denucleation takes place before the normal 
cells reach the blood. Just how we are to explain the disappearance of all 
trace of the nucleus from the normal erythrocyte is a question, but the general 
concensus of opinion seems to be that the nuclear material gradually fades 
within the cell, although some slight evidence of extrusion of the nucleus can 
be advanced: Although our ordinary methods show no such evidence, King^ 
has recently introduced a method by which he demonstrates stainable rem- 
nants of a nucleus in practically every red cell in the normal adult blood. 

Normoblasts (Trachyochromatic Erythroblasts). 

These are nucleated red cells similar in size, shape, and color to the 
normocytes. They do not usually show a biconcave form and do not unite in 
rouleaux. The protoplasm of the cell is usually regular in outline, stains more 
intensely than does that of the normocyte and frequently shows evidence of 
polychromatophilia, although it is normally orthochromatic.^ In myeloid 
leukemia, cells are frequently observed in which the protoplasm presents 
a ragged outline and may even, in some cases, be so degenerated as to show 
only a small fragment attached to the nucleus. These latter cells are prac- 
tically always polychromatophilic. This type of small cell might be called a 

1 Jour. Med. Research, igii, XXIV, 91. 

^ Certain small bodies (Mitochondria) of a lipoid nature are regularly present in nu- 
cleated red cells and in non-nucleated reds of the bone marrow as well as in non-nucleated 
forms in circulating blood of man in some diseases in which the blood forming organs are 
stimulated to increased activity. These bodies may be stained with Janus Green and other 
stains. See Cowdry, Internat. Monatschr. f. Anat. u. Physiol., 1914, XXXI, 267; Am. 
Jour. Anat., 1916, XIX, 423; Shipley, Fol. Haem., 1916, XX, 61; Sappington, Arch. Int. 
Med., 1918, XXI, 695; Cowdry, Carnegie Inst. Pub. 271, 1918, p. 39. 
35 



546 DIAGNOSTIC METHODS 

microblast, which is the rarest form of erythroblast and corresponds in size to 
the microcyte. Moreover, we may find such cells or fragments of cells 
attached to a nucleus, such cells corresponding in size and shape to the 
poikilocytes and being termed poikiloblasts. 

The nucleus of the mature normoblast (Howell's mature nucleated red) 
has a diameter of about one-third that of the cell, is densely stained, homo- 
geneous, sharply defined, spheroidal in shape, and without any decided 
chromatin network (the so-called pycnotic nucleus). It is situated rather 
toward the periphery than in the central portion of the cell and is surrounded 
by a clear zone shading off into the cellular protoplasm. Occasionally the 
nucleus is observed resting upon a margin of the red cell or even extruded 
entirely from it probably as a result of degeneration of the surrounding pro- 
toplasm (Pappenheim) . These nuclei often show amitotic figures, being 
subdivided into two or more lobes or fragments which may be connected by 
strands of chromatin. These mature forms may show all gradations in 
which the chromatin network becomes more and more evident, until we reach 
the very immature forms (Howell's immature nucleated reds). These latter 
cells are somewhat larger than the mature forms and are somewhat lighter in 
color; the nucleus is relatively larger and is composed of dehcate faintly basic 
chromatin fibers radially arranged, and frequently showing mitotic figures. 

These two types of cell are the forerunners of tne normocyte and their 
appearance in the adult blood is indicative of increased activity of the hemato- 
poietic organs, especially of the bone-marrow, either as a result of a poor 
condition of the blood itself or as a direct disease of the blood-forming organ. ^ 
These cells are most commonly seen in the milder forms of anemia, chlorosis, 
and acute anemia from hemorrhage, inanition, or organic disease. In the 
severe types of anemia they are constantly met with and are usually associated 
with the larger megaloblasts. During the course of severe anemia, especially 
in chlorosis, there obtains a periodic increase of normoblasts and of leucocytes 
lasting several days. This is followed by a marked increase in the normal 
number of red cells, giving rise to the condition described by von Noorden as 
a "blood crisis." The normoblasts disappear, the blood count falls off, and a 
second crisis may obtain. This condition is considered as a transitory at- 
tempt on the part of the bone-marrow to regenerate the blood. Such a crisis 
is not always a sign of improvement, but it does demonstrate that the type 
of blood formation is becoming physiological and that recovery may follow. 

Megaloblasts (Amblyochromatic Erythroblasts). 

These are cells of larger than normal dimensions, corresponding in size 
to the macrocytes and varying from 9 to 20 microns in diameter, if exceeding 
20 microns thay are known as the gigantoblasts of Ehrlich. Occasionally 
some cells are seen which are no larger than a normoblast, but in which the 
nucleus shows definite characteristics which should enable one to classify the 
cell as a megaloblast (Pappenheim). 

^See Weller, Jour. Med. Res., 1915, XXXIII, 271; Gilbert, Arch. Int. Med., 1917, 
XIX, 140; Drinker, Drinker and Kreutzmann, Jour. Exper. Med., 1918, XXVII, 249 
and 383. 



THE BLOOD 547 

The protoplasm of the megaloblast often appears swollen and enlarged 
(dropsical). The cell is usually circular or oval, but it is easily deformed, 
giving rise to irregular-shaped bodies. Although it usually contains an excess 
of hemoglobin, it may show a deficiency. It is usually polychromatophilic, 
the shade of cellular staining varying from yellow to purple. These various 
color tones may not be regular, but may be varied by tintings of almost any 
shade in the same cell. 

The nucleus of the megaloblast is very large, varying between 6 and 10 
microns. This may be situated either centrally or somewhat peripherally. 
It shows a great variety of forms appearing as a vesicular body with intra- 
nuclear network and nodal thickening, but without nucleoli. It may rarely 
be pycnotic, may show mitoses or many stages or karyorrhexis with frag- 
mentation, vacuolation, fading of the segments of the nuclei, as well as 
minute subdivisions into fine basic staining particles widely scattered in 
the cell (Ewing). It is frequently poorly defined and shows feeble basic 
staining qualities. It may be sharply differentiated from the body of the 
cell by a distinct white margin which is thrown into relief by the deeper 
staining of the nuclear and cell-substances. Occasionally the nucleus is over- 
looked owing to the polychromatophilic properties of the cell, which do 
not always permit of clear differentiation of nucleus and cell substance. 
Careful examination of the nuclear structure with its wide-open mesh- 
work, should, however, prevent the mistake of classifying this cell as a 
large lymphocyte. 

The clinical significance of the megaloblast is more or less in doubt. As 
such cells are foreign to the blood of an adult and as they are not present be- 
yond 15 per cent, of the total nucleated reds in the marrow, according to 
Emerson, it must be assumed that their presence in considerable number is 
indicative of a reversion to an embryonal type of blood formation, or at least 
denotes an arrested development of normal cells and in consequence an in- 
creased production of these abnormal types. Megaloblasts are, therefore, 
evidences of degeneration of blood-forming organs, while normoblasts are sig- 
nificant of regeneration of blood. A few of these cells, along with a larger num- 
ber of normoblasts, has no special significance in cases of severe anemia, being 
found in small numbers in any variety of anemia. If, however, the majority 
of the nucleated red cells be megaloblasts, especially if gigantoblasts be pres- 
ent and unequal mitoses be observed, a diagnosis of primary pernicious ane- 
mia seems justified. It may be considered that the number of erythroblasts 
has no special significance as regards the severity of a particular case, but that 
i1 indicates merely the effort on the part of the bone-marrow to overcome the 
effects of blood destruction. The appearance of megaloblasts may, therefore, 
be regarded as an evidence of incomplete formation of the younger elements 
and, not necessarily, as an unfavorable sign. In the anemia following infection 
with bothriocephalus latus, the specific toxins produce a megaloblastic de- 
generation of the bone-marrow, so that the blood picture may assume the 
characteristics of a severe pernicious anemia. Ehrlich and Lindenthal have 
reported a case of nitrobenzol poisoning, in the later stages of which the 



548 DIAGNOSTIC METHODS 

megaloblasts out-numbered the ery throblasts. Thayer^ is the first to report the 
finding of ameboid movements in a megaloblast in a case of Addisonian anemia. 
(D) Number of Red CeUs. 

The normal number of red cells is generally regarded as 5,000,000 per 
cmm. in the blood of the adult male, while the normal value for the female 
is 4,500,000. These figures are purely arbitrary, but they serve as an approxi- 
mate basis upon which one may form his opinion as to the probable normality 
of a specific specimen of blood. ^ We frequently observe marked variations 
in the red count under the influence of both physiologic and pathologic 
conditions. These variations may be due to an actual increase or decrease in 
the number of cells or to a change in the volume of the plasma as discussed 
under Total Volume of Blood on page 411. It is not uncommon to find much 
higher counts than these normal ones on healthy individuals, especially in 
those living the ''simple life," so that any special count must be a law unto 
itself and must be considered normal or abnormal only after considering all 
the factors which may influence the number of cells. 

Physiologic Variations. 
(i) Sex. 

The variations due to sex are not so marked as those from other causes. 
While the adult woman almost invariably shows a lower blood count than 
does the adult man, yet we occasionally find the girl showing, before puberty, 
a somewhat higher count than the boy of corresponding age, and also the 
woman, after the menopause, showing somewhat higher values than does her 
brother of similar years. It can hardly be doubted that menstruation, as 
well as pregnancy and lactation, have some influence in lowering a count in 
woman at certain periods of hfe, yet these conditions are simply transient and 
would not seem to have as much bearing on the question as does the somewhat 
more hydremic state of the normal plasma of woman. 
(2) Age. 

The number of the red cells varies more or less with the age of the subject 
examined. The highest values are usually observed at birth, when the count 
may run as high as 7,500,000, as in a case observed by the writer, the hemo- 
globin being practically always over 100 per cent, at this time; but, as a rule, 
a lower count is noted, averaging about 6,000,000. These high figures are 
due, to some extent, to the concentration of the plasma at birth through the 
loss of body fluid before a compensatory intake. The count gradually falls 
during the first few days and becomes fairly constant about the tenth day, 
when it is rare to find nucleated red cells. The number of cells is then stated 
to be somewhat reduced until the age of puberty, when a gradual increase 
occurs until about 40 years of age, after which a slight decrease may be ob- 
served in man. These variations, observed at different ages, are to be regarded 
as slight and influenced, to some extent, by the many modifications of the 
plasma as the result of growth and development as well as to the gradual 
decrease of functional activity after the middle periods of life. 

1 Arch. Int. Med., iqii, VII, 223; Ibid., 1911, VIII, 581. 
- See Bing, Ugesk. f. Laeger, 1919, LXXXI, 1483.. 



THE BLOOD 549 

(3) Altitude. 

An increase in the number of cells has been observed under the influence 
of a higher altitude. The count has been shown to increase at the rate of 
approximately 50,000 cells per 1,000 feet of ascent and to diminish, within 
36 hours, at a corresponding rate, this increase or decrease being more marked 
the more sudden the ascent or descent. Just what factors are at work in 
causing these changes is undecided.^ The rise is too rapid to be entirely 
accounted for by new formation of blood-cells under the influence of diminished 
oxygen tension, and the fall is not accompanied by signs of destruction of 
the reds. Weinzirl considers the increased count of high altitudes due to the 
lowered temperature at these elevations. This factor would seem to have 
some influence, as counts are frequently observed in which a variation is noted 
at places showing the same elevation. Change of residence from warm to a 
cold or from cold to a warm climate may lead to an increase or decrease,^ as 
the case may be, in the number of cells varying from 500,000 to 2,500,000. 

(4) Nutrition. 

The general nutritive condition of the subject has more or less influence 
upon the number of red cells per cmm. This statement must not be inter- 
preted to mean that the obese person shows a higher blood count than does 
his sparer brother. It is well known that obesity is an indication of poor as- 
similation of food; hence we should expect to find, as we really do, that the 
muscular person has a somewhat higher count than the obese subject and 
well-developed robust individuals a larger value than do the poorly nourished 
patients. Diet has, of course, much to do with the general nutrition, so we 
find the meat eaters averaging somewhat higher in their percentage of red 
cells than do vegetarians. Immediately following a hearty meal we may 
observe a temporary decrease in the number of red cells, but this soon returns 
to normal, owing to the rapid adjustment of the water content of the blood. ^ 

(5) Exercise. 

Active muscular exercise produces a transient increase in the red count due 
both to the increased blood-pressure and to the concentration of the blood 
through the loss of water by perspiration. Physical exercise, taken to the 
point of producing fatigue, may produce a marked diminution in the number 
of red cells, due probably to the fact that regeneration of new cells cannot 
keep up with the destruction of the old cells. Passive exercise, in the form of 
massage, has a transient influence in producing an increase in the number of 
the red cells, owing to its increasing the general circulatory tone ^ 

1 See Cohnheim, Kreglinger, Tobler, and Weber, Ztschr. f. physiol. Chem., 191 2, 
LXXVIII, 62; Schneider and Havens, Am. Jour. Physiol., 1915, XXXVI, 380; Dalhvig, Kolls 
and Loevenhart (Am. Jour. Physiol., 1915, XXXIX, 77) believe that a decrease in the oxygen 
tension of the respired air stimulates the bone marrow to increased production of erythro- 
cytes and hemoglobin. See Ocaranza, Gaceta Med. de Mex., 1919, I, 157; Gregg and 
Schneider, Am. Jour. Physiol., 1919, L, 216. 

2 Chamberlain has, however, shown (Philippine Jour, Sc, Sec. B., 191 1, VI, 467) that 
the average number of red cells in the tropics is little different from that in temperate 
zones. See Laquer, Deutsch. Arch. f. klin. Med., 1913, CX, 189; Cohnheim and Weber, 
Ibid., 225; and Lope, Semana Med., 1913, XX, 1077. 

3 See Hopmann, Arch. f. Verdauungskr., 1913, XIX, 456. 

^ See Hawk, Am. Jour. Physiol., 1904, X, 384; Schneider and Havens, Ibid., 1915 
XXXVI, 239. 



5 so DIAGNOSTIC METHODS 

(6) Baths. 

Careful investigation of the influences of both cold and hot baths has 
shown that an increase in the number of reds occurs under the action of these 
two conditions. The increase as a result of a cold bath may be as great as 
2,000,000 cells, due to capillary stasis as a result of vasomotor constriction. 
Likewise, a hot bath will increase the number of cells by causing dilatation 
of the peripheral vessels and a consequent increase in the amount of blood at 
the point from which the specimen is taken. When marked perspiration 
follows either a hot or cold bath the blood becomes concentrated and, as a 
result, an increase in the number of red cells will obtain. 

(7) Therapeutic Measures. 

Any drugs which cause rapid loss of fluid from the body, as for instance 
emetics, purgatives, diuretics, and diaphoretics, will cause concentration of 
the blood and hence a coincident increase in the number of cells, providing 
the change is sufficiently rapid and the blood examination is made before 
compensation occurs. Among the drugs which increase the number of red 
cells we find iron and arsenic, both of which are particularly valuable in 
anemic conditions, the former in chlorosis and the latter in pernicious anemia. 
The compounds of mercury and of lead, on the other hand, have a destructive 
action upon the red cells, so that we find these drugs causing a diminution in 
the number of cells. 

Pathologic Variations. 
(i) Oligocythemia. 

This is a condition characterized by a diminution in the number of red 
cells. It is usually associated with a decrease in the percentage of hemoglobin 
and with a slight reduction in the total volume of blood, although this latter 
factor is not invariably present. This condition is found, to a more or less 
degree, in all forms of anemia and may be temporary or permanent. The 
extent of the decrease in the number of red cells varies from 500,000 cells to 
a reduction of 4,000,000. This diminution in the number of cells is usually 
an indication of the severity of the anemia, the most marked decrease being 
observed in the pernicious types, Osier reporting probably the lowest count 
recorded, namely, 100,000 red ceils. 

While this oligocythemia is usually associated with oligochromemia, yet 
we find in chlorosis that the diminution of red cells is not as marked as is the 
reduction in the amount of hemoglobin, the color index in this condition being 
usually low. In some cases of chlorosis, however, the oligochromemia may 
keep pace with the oligocythemia. In pernicious anemia, on the other hand, 
we have a marked reduction in the number of cells and coincidently a large 
decrease in the amount of hemoglobin, the result being that we have a very 
high color index as the usual sign of this condition. Cases showing the loss 
of a large amount of blood, as the result of hemorrhage have naturally an 
oligocythemia, but the percentage of hemoglobin may not necessarily be 
reduced to any great extent. This gives rise to the condition of secondary 
anemia, in which the color index may be high. In these cases of hemorrhage 



THE BLOOD 55 1 

a sudden reduction to the point of 1,000,000 or less cells is usually followed 
by a fatal result, although even here recovery has been made possible by 
rapid infusion of salt solution and compensatory activity of the blood-form- 
ing organs. In leukemia we find that the red cells are not usually diminished 
to a very great extent, the oligocythemia being generally more marked in the 
lymphatic than in the myeloid variety. Occasionally, however, we do find 
very low counts, in both varieties of leukemia. These same statements 
apply to splenic anemia, although the count here very rarely reaches a lower 
point than 2,500,000 cells. 

A large number of conditions, aside from direct blood diseases, cause a 
diminution in the number of these red cells. Thus we find that the toxins 
of certain of the specific fevers, such as typhoid and pneumonia, may cause 
a marked anemia, but this is not necessarily the rule. Acute infection with 
pus organisms is frequently observed to cause an extreme and rapidly pro- 
gressive anemia, the destruction of the red cells being in some cases very 
extensive. In malignant disease we usually find, especially if cachexia is 
present, a very extensive anemia which may lead to a diagnosis of the per- 
nicious type. It is to be remembered, however, that a secondary anemia may 
assume all of the characteristics of the primary pernicious type. 
(2) Polycythemia. 

This is a condition characterized by an increase in the number of red cells 
and is sometimes called polyglobulia.^ Whether this increase is actual and 
permanent or whether it be simply apparent, due to concentration of the 
blood or to unequ^al distribution of blood in the peripheral vessels, is still an 
unsettled question. It is true that many physiological causes previously enu- 
merated do bring on a polycythemia, so that these should be remembered when- 
ever a blood count is to be made. From the pathologic standpoint \ve find 
an increase in the number of red cells occasionally following active blood 
regeneration after hemorrhage. Likewise, we observe an increase in the 
number of cells in phosphorus poisoning, acute yellow atrophy of the liver, 
and in certain cases of general hepatic insufficiency. Why such an increase of 
cells occurs in these conditions is very hard to say. It has been found that 
the solids of the plasma are not increased, so that we are not warranted in 
assuming a concentration of the fluid elements. A polycythemia of uncer- 
tain origin is also seen in cases of poisoning with carbon monoxid and illumi- 
nating gas.^ 

In a condition called by Osier autotoxic enterogenous cyanosis, which is 
characterized by a marked increase in the number of red cells and also by 
enlargement of the spleen, we have reason to assume that a direct real increase 
in the number of red cells occurs, but the pathogenesis of the condition is 
uncertain. It has been traced to improper aeration of the blood and occurs 
also in congenital heart disease, mitral lesions in the adult, and in pneumonia 
and acute miliary tuberculosis . While this high blood count must be definitely 

^ Also erythremia. 

2 See Karasek, Trans. Chic. Pathol. Soc, 1911, VIII, 173; Lipsitz and Cross (Arch. 
Int. Med., 191 7, XX, 889) and Lipsitz, Fuerth and Cross (Ibid., 913) report a count of 
10 million reds in a case of poisoning with cantharides. 



552 DIAGNOSTIC METHODS 

admitted, its importance from a clinical standpoint is still unsettled. It 
is uncertain whether the rapid increase in the number of reds is due to an 
attempt on the part of the blood-forming organs to overcome the influences 
of the toxic substances upon proper oxidation in the system; yet it is certain 
that relief very frequently follows such an increase in the number of red cells, 
although in Osier's disease or, as it is called by some, Vaquez's disease, death 
frequently follows without any relief from the condition.^ 
(E) Staining Properties of the Red Cells. 

The normal red cell, like all living cells, is incapable of being stained with 
anilin dyes; that is, it is achromatophilic. Previous to staining, fixation of 
the cell must take place, the protoplasm being killed in this process. The 
normal fixed red cell has a marked affinity for various dyes of the acid type, 
such as eosin, orange-G, acid fuchsin, and indulin, and is therefore called 
acidophilic or oxyphilic. This cell normally takes up but one color from a 
mixture of dyes, and is called, therefore, monochromatophilic. 

Polychromatophilia. 

Under various pathologic conditions we find red cells which show a tend- 
ency to take up the basic stains, and for this reason are called basophilic or 
polychromatophilic cells. The tint of such abnormal red cells varies from 
that of a light indefinite shade of the basic stain to a dark distinct tone. Just 
what factors are at the bottom of this change, which is called polychromato- 
philia or polychromasia, is difficult to say. The normal acidophilic tenden- 
cies of the cell are due to the presence of hemoglobin and, in consequence, the 
normal staining properties will depend upon the relative richness of the cell 
in this pigment. As hemoglobin is always acidophilic, we cannot assume 
that the polychromatophilic properties of the abnormal red cells are due to 
variations in the hemoglobin, but must content ourselves with the belief that 
these changes have something to do with the protoplasm of the cell.^ 

This condition, which has been termed by Ehrlich anemic degeneration, 
is characterized by a diffuse basic staining property of the red cells. These 
basophilic cells are somewhat larger than the normal ones, show less bicon- 
cavity, and often are abnormal in shape. The megaloblasts are practically 
always polychromatophilic and the normal erythroblasts of the bone-marrow 
usually show this degeneration. This fact would seem to point to the prob- 
ability of this condition being a sign of regeneration of the blood as the ery- 
throblasts are more prone to be polychromatophilic the younger they are. 

Two distinct forms of polychromasia have been found. The first of these, 
known as the polychromatophilic degeneration of G abr its chew sky , is the diffuse 
basophilic staining of the cells which is found in various forms of anemia and 

1 See White, Lancet, 1912, XCII, 7; Lucas, Arch. Int. Med., 1912, X, 597; Cahn, Inaug. 
Dissert., Berlin, 191 2; Chauffard and Troisier, Presse med., 1913, XXI, 653; Friedman, 
Med. Record, 1913, LXXIV, 701; and Moewes., Deutsch. Arch. f. klin. Med., CXI, 281; 
Hertz and Ehrlich, Deutsch. Arch. f. klin. Med., 1914, CXVI, 43; Mosse, Ztschr. f. klin. 
Med., 1914, LXXIX, 431; Lamson, Jour. Pharmacol, and Exper. Therap., 1915, VTI, 169; 
Lamson, Jour. Pharm. and Exper. Ther., 1Q15, VIT, 169; Ibid., 1916, VITI, 167; Lamson 
and Keith, Ibid., 247; Freund and Rexford, Arch. Int. Med., 1916, XVII, 415; Pickard, 
Jour. A. M. A., 1916, LXVII, 1845; Lankhout, Nederl. Tijdschr. v. Geneesk., 1917, T, 294; 
Goyena and Masoch, Sem., Med., 1919, XXVI, 113. 

^ See Montagnani, Sperimentale, 1919, LXXIII, 21. 



THE BLOOD 553 

extensively in the cells of the normal bone-marrow. The second of these, the 
polychromasia of Maragliano, is shown in severe anemias and in toxemias and 
appears as a more punctate basophilia, being closely related to the basophilic 
degeneration of Grawitz, which will be discussed later. While the poly- 
chromatophilia of the diffuse type is considered by Ehrlich as evidence of a 
degenerative process, that is as a coagulation necrosis of the discoplasm in 
consequence of which this takes up the albuminous principles of the plasma 
while it loses its power of retaining hemoglobin, yet there are evidences show- 
ing that this may be a regenerative process.^ Polychromatophilia is often 
seen in cells which are undergoing degeneration, especially those following 
malignant disease, eruptive fevers, malaria, and after various poisons, such 
as that of snake venom. 

The forms of partial polychromatophilia, which have been described 
under various names, such as vacuolization, pseudonucleation, globular 
decolorization, and more commonly Maragliano 's endoglobular degeneration, 
are seen in normal blood in from 30 to 70 minutes after the specimen is made. 
They are usually found in the center of the cell, but may be near the periphery. 
There may be several such areas in a single cell, but the more common form is 
the single area of degeneration, which is usually round, may be elHptical, and 
may resemble a vacuole. These are shown quite distinctly in the unstained 
specimen and in this condition very frequently are seen in rapid motion. 
This motion is not of the true ameboid type, but is due more to the gradually 
progressing coagulation and consequent constriction of the protoplasm. 
These areas, whether in the fresh or stained specimens, may be mistaken for 
malarial parasites, so that the worker should be on his guard lest he make a 
wrong diagnosis without sufficient evidence. They differ in size from the 
parasites, and on focusing the specimen are characterized, usually, by a 
change from a smaller to a larger form, which variation is not evident with the 
malarial organism. The longer one searches in the unstained specimen for 
malarial organisms the more apt a mistake in diagnosis is to be made, as these 
'^Maraglianos" develop quite rapidly. In the stained specimen these areas 
of coagulation necrosis will show a basophilic staining quality, while the 
chromatin element of the malarial organism will differentiate this body from 
the more usual degenerative area.. 

(F) Degenerations. 
While the preceding conditions of polychromatophilia are to a certain 
extent true degenerations, yet they are more properly grouped under the 
heading of atypical staining reactions of the red cell. 

Basophilic Degeneration of the Red Cells. 

This condition is distinct from the polychromatophilia above described 
and is known as the punctate basophilia of Grawitz. It is characterized by the 
appearance, in the body of the red cell, of granules of varying size, which 
stain with the basic dyes. The cell may be dotted throughout with these 
granules or may show this degeneration only in parts. These granules are 

' See Pfuhl, Ztschr. f. klin. Med., 1913, LXXVIII, 102. 



554 DIAGNOSTIC METHODS 

not observed in the fresh unstained specimen and are not increased, as are the 
Maragliano areas, by allowing the blood to stand. These cells are observed 
in pernicious anemias, leukemia, in the toxemia of malignant states and 
especially in cases of lead-poisoning. 

The size of the granules may vary from small dots to large granules, 
showing a diameter of one or more microns. Their origin is much in doubt, 
but they are generally considered to be areas of coagulation necrosis associated 
with either degeneration or regeneration of the red cells. They are quite 
distinct from the granular basic "stippling" observed in malarial conditions, 
but are probably evidences of direct toxic effects upon the protoplasm of the 
red cells. Their greatest clinical importance is probably in lead-poisoning, 
where they may be the only signs of abnormality in the blood picture. They 
may vary in number from day to day, showing in some cases five or six in one 
microscopic field, but, as a rule, they are present in much fewer numbers, as 
one observes them only after examining several visual fields. As a rule, they 
appear very early in cases of lead-poisoning, in one case observed by the 
writer within three days, and they may be present in the blood of a lead 
worker for several years after his exposure to the effects of the lead. They 
are usually the first sign of anemic change and usually persist longer than do 
the other abnormalities of the blood. ^ 

Ring Bodies. 

Occasionally one observes in the red cells curious ring-like bodies which 
are in shape very much like the hyaline malarial ring with a circular refractive 
center. They change their shape in a peculiar way, much resembling the 
undulatory movements of the hyaline body. They do not increase in number 
or grow larger on standing as do the Maraglianos and they are observed in 
a large number of conditions, such as measles, pernicious anemia, and severe 
secondary anemia. Two types of these ring bodies must be distinguished, 
the first is usually more peripherally situated, occasionally has a definite 
crescentic shape or has an appearance much resembling that of a Maragliano, 
and is found especially in cases of measles. This finding has lead to the 
assumption that these bodies were the organisms causing measles. The 
second form has been described by Cabot^ and more recently by Schleip^ in 
cases of pernicious anemia, leukemia, and secondary anemias. These latter 
bodies are larger than the former, more distinct, irregular in shape, frequently 
forming figure-eight bodies and are usually stained bright red with Wright's 
stain, but may occasionally show a blue shading. Herrick^ has observed 
thin, elongated, sickle-shaped red cells, some of them nucleated, in a case of 
severe anemia. 

Various other forms of degeneration of the red cells occur, as for instance 

^ See Petry, Biochem. Ztschr., 1912, XXXVIII, 92; Anders, Jour. Am. Med. Assn., 
1914, LXII, 1164; Linenthal, Ibid., 1796; Schnitter, Deutsch. Arch. f. klin. Med., 1915, 
CXVII, 127; Moller, Hospitalstid., 1915, LVIII, 1287 and 1303. 

2 Jour. Med. Research', 1903, IX, 15. 

2 Deutsch. Archiv. f. klin. Med., 1907, XCI, 449. 

* Arch. Int. Med., 1910, VI, 175. See, also, Bishop, Arch. Int. Med., 1914, XIV, 388; 
Cook and Meyer, Ibid., 1915, XVI, 644; Pisani, Folia Haemat., 1915, XIX (i), 119; Emmel, 
Arch. Int. Med., 191 7, XX, 586. 



PLATE XIX. 



fO 







.-ft; 



e.d. 



^ 



Xa1Vla^.r.f Kiil 



Ring Bodies in Red Cells. (After Schleip.) 



THE BLOOD 555 

the appearance of rod-like areas resembling bacilli/ which may keep up a con- 
stant vibratory motion carrying them through the entire substance of the 
cell. This finding should not confuse one in making a diagnosis of the 
presence of bacteria. Another form of degeneration, known as Ehrlich's 
hemoglobinemic degeneration, has the appearance of a small dark cell lying 
upon a larger paler one. These are probably areas of condensed protoplasm 
with the hemoglobin distinctly separated from the stroma. Such cells 
appear, occasionally, in certain types of malaria and are shown as corpuscles 
in which the hemoglobin is apparently condensed around the parasite. This 
degeneration is occasionally seen in nucleated red cells and may give the 
appearance of a microblast lying upon a macrocyte. It is best seen in cases 
of pernicious anemia and may explain some of the ''acidophilic granules" of 
the red cells which have been described (Emerson). 
(G) Isotonicity and Resistance of the Red Cells. 

Normally, the relations of the cellular to the fluid portions of the blood 
are such that the hemoglobin and other constituents of the red cells are held 
intact within the limiting membrane of the cell. Any change in the osmotic 
pressure of the plasma is certain to be manifested by variations both in size 
and in composition of the cellular elements. We find, therefore, as previously 
discussed under the heading of Osmotic Pressure, that the inorganic constitu- 
ents of the plasma are the factors upon which the volume of the red cell 
depends to a large extent. 

By an isotonic solution, as applied to the blood, we mean a solution of 
such a strength as to preserve the corpuscles and to prevent passage of water, 
salts, and organic bodies from the plasma into the corpuscle or from the cor- 
puscle into the plasma. In other words, an isotonic solution is one whose 
osmotic pressure is equal to that of the contents of the red cell. While the 
limiting structure of the red cell is indefinitely known, it seems to have certain 
properties which would lead us to assume that it is a semipermeable mem- 
brane, that is one which will permit the passage of organic compounds or of 
salts or their ions in both directions. This apparent selective activity is 
dependent both upon the laws of osmosis and diffusion through a semiper- 
meable membrane and upon the basic ideas of Ehrlich's side-chain theory. 
While other figures have been used at various times to represent the strength 
of a solution isotonic with the red blood-corpuscle, the one which seems more 
nearly to duplicate the actual condition is a 0.9 per cent, solution of sodium 
chlorid. This is or should be the normal salt solution which is used in physi- 
ological work and in transfusion. Loeb has shown that a properly balanced 
solution containing the chlorids of sodium, potassium, and calcium more 
nearly represents a proper "physiological salt solution" than does the simple 
0.9 per cent, sodium chlorid solution. Such an isotonic solution will preserve 
the corpuscles, will prevent the passage of hemoglobin from the cells into the 
plasma, and will not permit of shrinkage or swelling of the cells. ^ Solutions 

^ See Roth, Ztschr.f. klin. Med., 1912, LXXVI, 23. 

2 Ringer's solution is frequently used in this work. Its composition is : sodium chlorid, 9 
grams; calcium chlorid, 0.24 gram; potassium chlorid, 0.42 gram; sodium bicarbonate, 0.2 
gram; water, 1,000 c.c. 



556 DIAGNOSTIC METHODS 

which are stronger than the one above mentioned are called hypertonic 
solutions, while those of a lower concentration are termed hypotonic solutions. 
In the first of these shrinkage of the cells will occur, while in the latter phe- 
nomena of swelling will be observed. 

The erythrocyte normally shows a certain amount of resistance to varia- 
tions in the osmotic pressure of the blood and to the hemolyzing effects of 
various substances. It is true that the osmotic pressure of the plasma may 
be raised by the introduction of certain substances into the blood without 
causing phenomena of shrinking, swelling, or hemolysis to occur, yet we must 
not assume that the cell is not influenced by such changes in osmotic pressure. 
Simple variations in osmotic pressure come more nearly to the foreground in 
the explanation of these changes, especially of those occurring in most dis- 
ease processes of a chronic type, while in the more acute infectious types 
associated with toxemia the biologic theory must be invoked. As yet little 
of value has been forthcoming from the study of the resistance of the blood 
cells to variation in osmotic pressure, as the scope of the examination has not 
been sufficiently widened by application of accurate experimental methods. 
The writer is forced to say, from the results of his own experiments, as well as 
those of others with the more usual method of Hamburger, that no reliable 
data are at hand. It is highly probable that the little understood biologic 
influences are clinically much more important than are those due to variations 
in osmotic pressure, as this latter process cannot explain the hemolytic results 
of the toxins or of certain definite chemical compounds which bring about 
these results in concentrations which have little influence upon the tonicity of 
the blood. ^ 
(H) Variations of the Red Cells in Childhood and in Old Age. 

As previously stated, the average number of red cells is much higher than 
the normal figure at birth, gradually decreasing until the age of puberty 
when a gradual increase occurs again until middle life, after which a steady 
decrease is observed. It is to be expected that variations in the color tones 
(hemoglobin content) of these cells will vary normally with the age of the 
subject, being very high in the early days of life, gradually diminishing as 
puberty approaches, increasing until the middle periods of life, and steadily 
declining as the age increases. Aside from the number of cells, the blood in 
childhood shows little variation in the characteristics of the red cells, but the 
variations in the plasma are more or less marked, as the synthetic and retro- 
grade products of tissue metabolism are much more evident in the period of 
development than during the middle periods of life. 

As the subject becomes older the blood is laden with products of retrograde 

tissue change and the blood-forming organs become less active. It must be 

remembered that the various organs of the body contribute certain factors to 

^ See Pepper and Peet, Arch. Int. Med., 1913, XII, 81; also, Gaisbock, Deutsch. Arch. f. 
klin. Med., 1913, CX, 413; Holler, Ztschr. f. klin. Med., i9i4,LXXXI, 129; Hober, Deutsch 
med. Wchnschr., 1915, XLT, 273; Roccavilla, Rif. Med., 1915, XXXI, 813 and 873; Ham- 
burger, Biochem. Ztschr., 1915, LXXI, 464; Hill, Arch. Int. Med., 1915, XVI, 809; Loeb, 
Jour. Biol. Chem., 1916, XXVII, 339, 353, and 363; Hamburger, Wien. Med. Wchnschr., 
1916, LXVI, 521 and 575; Davis, 111. Med. Jour., 1916, XXIX, 280; Musser and Krum- 
bhaar. Jour. A. M. A., 1916, LXVII, 1894. 



THE BLOOD 557 

the blood and may, therefore, all be considered as blood-forming organs in a 
definite sense. The ductless glands, in particular, pour into the blood certain 
substances which markedly influence the proper correlation of these various 
organs. Just what these substances are is not well established, so that the 
introduction of the word "hormone" by Starling does not at present have a 
specific meaning. As old age approaches variations become evident in the 
plasma, and through such influences changes in the morphology of the red 
cells are more or less frequent. Besides these plasma changes, gradual 
atrophic degeneration of the blood-forming organs takes place, so that we 
may find red cells showing the various types of anemic degeneration and may 
even see nucleated red cells as an evidence of the attempt on the part of the 
bone-marrow to overcome the gradual destruction of cells. The more nota- 
ble variations in the red cells are observed in their size, owing to the hydre- 
mic state which is almost always characteristic of the plasma in advanced 
periods of life, but associated diseased conditions will, of course, give their 
characteristic changes. 

(7) Functions of the Red Blood-cells. 

The only well-recognized function of these cells is their oxygen-carrying 
power, which is dependent upon their hemoglobin content. Other oxygen 
catalysts, such as oxidase, peroxidase, and catalase, are found more largely in 
the leucocytes and play a significant role in the function of the whole blood. 
These will be discussed later. As is well known, the fluid portions of the 
blood are much more influenced by the various organs of the body than are 
the cellular elements, although the red cells will naturally be affected by 
variations in the hematopoietic organs, such as the bone-marrow, liver, and 
spleen. Our knowledge regarding the variation in the plasma is so uncertain 
that we have been led to interpret the changes in the histological appearance 
of the blood as significant of certain general pathological conditions. It is 
true that in some cases we do have direct disease of the blood-forming organs 
and, in consequence, variations in the cellular structures are of diagnostic 
importance, yet in the large majority of cases the preliminary changes must 
be resident in the plasma, those in the cellular structure being simply inci- 
dental. It may be, therefore, necessary to assume a functional activity on 
the part of the red cells to overcome biologic changes in the plasma and if so 
we must attempt to discover just what plasma changes we are to regard as 
significant of the various conditions shown by the red cells in each true blood 
disease.^ 

(6) Leucocytes. 
(A) Appearance. 

In the fresh specimen the leucocytes appear as colorless, high refractive 
bodies, somewhat larger than the red cells, and showing a definite nucleus. 
These white cells show distinct ameboid movement in the fresh specimen by 

^ Rous and Robertson (Jour. Exper. Med., 1917, XXV, 651 and 665) show that the nor- 
mal fate of the erythrocytes, in those species in which phagocytosis is negligible, is to be 
fragmented and removed from the blood by the spleen, and, under exceptional circum- 
stances, by the bone marrow. 



558 DIAGNOSTIC METHODS 

virtue of which they are able to surround a foreign body and enclose it within 
their own protoplasm. This is the well-known property of phagocytosis 
which is such an important factor in Metschnikoff's theory of immunity. In 
contradistinction to the red cells, these white cells show many variations both 
in size and in shape. As the relations between the nucleus and the cellular 
protoplasm is quite distinct in many of these forms, some writers have been 
led to classify the white cells according to the peculiarities of the nucleus. 
Other writers classify them into granular and nongranular forms, as many of 
these cells show distinct granulations which are more clearly differentiated in 
the stained specimens. As more or less confusion exists regarding these 
different variations, it seems more rational at present still to classify these 
white cells with Ehrlich, who combines both the nuclear and granular char- 
acteristics in his classification.^ 

{B) Leucocytes in Normal Blood. 

(i) Lymphocytes. 

The cells of this class which occur normally in the blood are of two kinds, 
(a) the small cell, about 5 to 8 microns in diameter and (b) large cells showing 
a diameter of from 8 to 10 microns. Both of these cells have a very large 
nucleus which is usually centrally located but may have an eccentric posi- 
tion. It may be compact or coarsely reticulated and is not always as strongly 
stained as is the surrounding cytoplasm. Nor is it as deeply stained as is 
the nucleus of the normoblast, although it is very rich in chromatin. The 
nucleus of the larger cell may show very irregular staining properties. The 
nuclei of both of these cells are usually circular in outline, but may show an 
oval or a kidney-shaped structure, in which at times may be seen a distinct 
nucleolus. The protoplasm of these cells is usually in the form of a narrow 
rim surrounding the nucleus and showing a strongly basophilic homogeneous 
cytoplasm. The older cells may at times show a difficult staining property, 
may even be acidophilic, and occasionally exhibit a net-like structure in which 
are observed a few granules scattered throughout the protoplasm of the cell. 
This granulation, which occurs in about one-third of the cells seems to have 
little clinical significance, except as a probable sign of age of the cell, this 
granular stage being regarded as the end-point of their development. These 
granules are probably not representatives of true granulation, but seem to be 
more referable to nodal points of the reticular structure of the protoplasm.^ 

These cells usually constitute from 20 to 25 per cent, of the leucocytes, 
their absolute numbers being from 1,200 to 2,000 per cmm., but the relative 
proportion of the small and large form being unsettled. The larger types of 
these cells are more closely associated with increased functional activity of the 
lymphoid tissues and should probably be considered the older cell of the 
two. According to Pappenheim, the large lymphocyte represents the mother 
cell from which all other leucocytes as well as erythrocytes are indirectly de- 

^ See Pappenheim, Die Ergebnisse derinn. Med. u. d. Kinderheilkde. 1913, VIII, 183; 
also, Hoxie, Interstate Med. Jour., 1913, XX,, 1049. 

2 See Pappenheimer, Jour. Exper. Med., 1917, XXV, 633; Ibid., XXVI, 163; Mcjunkin, 
Arch. Int. Med., 1918, XXI, 59. 



DESCRIPTION OF PLATE XX. 



{Trtacid Stain.) 

1, 2, 3, 4. Small Lymphocytes. 

Contrast the faintly colored protoplasm of these cells in the triple stained specimen 
with their intensely basic protoplasm in the film stained with eosin and methylene- 
blue, 17 and 18. The cell body of i is invisible. Note the kidney-shaped nucleus in 4. 

5, 6. Large Lymphocytes. 

With this stain the nucleus reacts more strongly than the protoplasm: with eosin 
and methylene-blue (19, 20), on the contrary, the protoplasm is so deeply stained 
that the nucleus appears pale by contrast. This peculiarity is also observed in the 
smaller forms of lymphocytes. 

7, 8. Transitional Forms. 

Note the moderately basic and indented nucleus, and the almost hyaline non- 
granular protoplasm. Compare 8 with the myelocyte, 7, Plate IV, these cells dif- 
fering chiefly in that the myelocyte contains neutrophile granules. 

9,10,11. Polynuclear Neutrophiles. 

These cells are characterized by a polymorphous or polynuclear nucleus, sur- 
rounded by a cell body filled with fine neutrophile granules. In 11 the nuclear 
structure is obviously separated into four parts; in 9 it is moderately, and in 10 
markedly, polymorphous. 

12, 13. Eosinophiles. 

The nuclei are not unlike those of the polynuclear neutrophile, except that they are 
somewhat less convoluted, and poorer in chromatin, staining less intensely. The 
protoplasm is filled with coarse eosinophile granules, the characteristics of which 
are clearly illustrated by 13, a " fractured " eosinophile. 

14. Eosinophilic Myelocyte. 

Compare with 15. 

15, 16. Myelocytes. {Neutrophilic.) 

These cells are morphologically similar to 14, except that they contain neutrophile 
instead of eosinophile granules. Note that the granules of the myelocyte are 
identical with those of the polynuclear neutrophile. A dwarf form of myelocyte is 
represented by 16. 

{Eosin and Methylene-blue.) 

17, 18. Small Lymphocytes. 

Note the narrow rim of pseudo-granular basic protoplasm surrounding the nucleus 

and the pale appearance of the latter. 
19, 20. Large Lymphocytes. 

Budding of the basic zone of protoplasm is represented by 20, Both of these cells 

belong to the same type as 5 and 6. 
21, 22. Large Mononuclear Leucocytes. 

Compared with 19 and 20, these cells have a decidedly less basic protoplasm, but a 

somewhat more basic nucleus. In the triple stained film these differences cannot 

be detected, so that they must be classed as large lymphocytes. 
23. Transitional Form. 

The distinction between this cell and 24 is not marked; the nucleus of the latter 

simply being somewhat more basic and convoluted. 
24,25,26,27. Polynuclear Neutrophiles. 

With this stain these cells show a feebly acid protoplasm, and lack granules. Note 

that the more twisted the nucleus the deeper it is stained. Compare with 9, 10, 

and II. 
28, 29. Eosinophiles. 

Compare with 12 and 13. 

30. Eosinophilic Myelocyte. 

Compare with 14. 

31. BasDphile. {Finely granular.) 

This cell is characterized by the presence of exceedingly fine 5-granules, staining 
\.\i&pure color of the basic dye. The nucleus is markedly convoluted and deficient 
in chromatin. The cell here shown was found in normal blood. 
32,33,34,35,36. Mast Cells. 

The granules take a modified basic color, as shown by their royal-purple tint in this 
illustration. Note their unusually large size and ovoid shade in 35, their peculiar 
distribution in 35 and 36, and their irregularity in size in 32 and 36. With the triacid 
mixture these granules, as well as those of the finely granular basophile, 31. remain 
unstained, showing as dull-white stippled areas in the cell body. The nuclear chro- 
matin of the mast cell is so delicate and so freely stained that it is barely visible. 
These cells were found in the blood of a case of spleno-medullary leukemia. 



, PLATE XX. 



1 ^ 



/M 




10, 



W%./.4?^ 



14 







13 






17 18 



0( 



23 



,C>^- 



31 



% 




'i^lrf*** 



The Leucocytes. 

(i-i6, Triacid Stain ; i']-2,6, Eosin and Methylene-blue.) 

(E. F. Faber, /^c.) 

(From £)a Costa's ''Clinical Hematology.'") 



THE BLOOD 559 

veloped as a result of heteroplastic differentiation. Variations in technic of 
preparing the slides will often lead to differences in size of these cells, the 
thinner preparations showing these cells as larger forms, while the thicker 
preparations present the smaller type of these cells. 

These cells are beautifully colored by the hematoxyhn-eosin stain and by 
the various modifications of the Romanowsky stain, although the boundary 
between the nucleus and the protoplasm is not always clearly outlined by 
these latter stains. The granules, which are occasionally found in these cells 
and which are in size between the a and the € granules of Ehrlich, show most 
clearly in preparations stained by the Giemsa stain and appear distinctly 
azurophilic. These granules do not appear at all with the triple stain. 

These cells are frequently increased and frequently diminished, the 
former condition being spoken of as lymphocytosis, the latter as lymphopenia. 
A relative increase of the number of these lymphocytes is regarded as more or 
less characteristic of typhoid fever, especially when associated with a diminu- 
tion in the total number of white cells. In lymphatic leukemia these cells are 
present in large numbers and are indicative of marked disturbance in the 
lymphatic structures. 

(2) Large Mononuclear Leucocjrtes. 

These cells, which are supposed to be derived from the spleen and are 
called, therefore, splenocytes, are as a rule two or three times as large as a 
red cell (12 to 15 microns), and show a nucleus which may be large and round, 
but is more frequently oval in shape or may be indented, forming the so- 
called kidney type of nucleus. It is to be remembered that we may have both 
large and small types of this large mononuclear leucocyte, the smaller forms 
being distinguished from the lymphocyte by the relation of the protoplasm 
to the nucleus. These nuclei are usually eccentric in position and are not 
always sharply outlined, they are poor in chromatin, but are strongly baso- 
philic, although less so than are the nuclei of the lymphocytes. The proto- 
plasm of these cells is very abundant in relation to the size of the nucleus and is 
very clear, hyahne, and nongranular in appearance. This protoplasm is 
much less basophilic than is the nucleus and shows a very fine reticulum with 
nodal thickenings which are somewhat more strongly basophilic and may give 
the appearance of granulation.^ These cells should not, however, be regarded 
as granular cells. 

Occasionally we find cells, derived from the above, which have been called 
by Ehrlich "transition forms," which are the largest of all the white cells. ^ 
The nucleus is pale, often deeply notched giving it usually the appearance of 
a saddle-bag, and shows the characteristics of the large mononuclear type, 
being distinguished from the polymorphous nucleus of the neutrophile cells 
by its greater thickness and by its diminished intensity of staining. The 
extensive granulation of the polymorphonuclear leucocyte should make any 
mistake in diagnosis impossible The protoplasm of these transitional cells 

^Bergel (Deutsch. Arch. f. klin. Med., 1912, CVI, 47) has shown that these cells 
play a large role in the hemolysis and lipolysis noted after injection of cells and lipoids. 
2 See Evans, Arch. Int. Med., 1916, XVII, i; Ibid., XVIII, 692. 



560 DIAGNOSTIC METHODS 

is very abundant and shows faint basophilic properties and may even have 
a few fine granules, which are neutrophilic in character, in the neighborhood 
of the nucleus. These two forms of cell constitute between 3 and 5 per cent, 
of the leucocytes, their absolute number varying between 200 and 400 per 
cmm, Pappenheim considers that these large mononuclear cells develop 
directly from the large lymphocytes and then pass into the transition forms, 
which are the final developmental types. 

(3) Polymorphonuclear Cells. 

(a) Polymorphonuclear Neutrophiles. 
These cells, sometimes called the finely granular cells of Schultze, are 
from two to three times the size of the red cell (10 to 12 microns) in diameter, 
the size depending upon the extent to which the cell is flattened by pressure. 
These cells are always smaller than the large mononuclear type and quite 
small specimens are frequently found in cases of myeloid leukemia. They 
are the most sharply characterized cells of the blood, their protoplasm being 
relatively great and showing slightly acidophilic staining properties. The re- 
ticular portion of the protoplasm is very slightly basophilic, showing occasion- 
ally nodal thickening or granules when stained with the methylene blue 
dyes. Throughout this protoplasm are scattered fine dust-like granules, the 
€ granules of Ehrlich, which are not all of the same size and which stain with 
the neutral principle of the dyes. In some cases these granules may gradu- 
ally diminish so that they may be apparently absent or at least undetected. 
The triple stain is the characteristic stain for these granules, showing them 
of a distinct lilac color; but various other acid dyes, such as eosin, give 
various tints to them, so that they are. recognizable as distinctly reddish 
granules in specimens stained by any of the Romanowsky modifications. 
The nuclei are elongated and constricted and may appear in the form of a 
bent rod or a mass of interwoven fine fibers, showing a thin chromatin-rich 
structure, or a distinctly S-shaped formation with oval thickenings. Fre- 
quently these nuclear masses appear as if separated into two or more distinct 
nuclei, but as a rule these masses are connected by fine bands and should, 
therefore, be considered rather as polymorphous than as polynuclear. The 
nucleus shows a reticular structure with nodal thickenings and is very baso- 
philic. These forms constitute from 65 to 75 per cent, of the total number of 
cells, their absolute number averaging 5,000 per cmm., the percentage rang- 
ing from 20 to 40 per cent, in the first years of life when the lymphocytes are 
increased. When outside of the blood-vessels they form the usual pus-cells 
and are extremely active phagocytes. These cells are derived from the mono- 
nuclear neutrophilic myelocytes which are normal habitants of the bone- 
marrow. They run their course as such and are not transformed into other 
cells, their granules diminishing in pathological conditions or with age, 
such change being associated with degeneration in the nucleus. 

Perinuclear Granulation. 

Sometime ago Neusser reported a finding of basophilic granules in 
certain leucocytes, especially the mononuclear and polynuclear type. These 



THE BLOOD 56 1 

granules surrounded the nucleus and even appeared attached to it, showing 
a variable size and being more or less refractive. He regarded these perinu- 
clear granulations as characteristic of the uric acid diathesis, but the work 
of Futcher and of Simon show that these granules are undoubtedly artefacts 
which can be produced by heating and by variation in the staining. 

Ameth's Classification. 

Arneth^ in his study of the blood in health and disease has been led 
to classify the polymorphonuclear neutrophiles into five classes depending 
upon the number of nuclear lobes. His divisions seem to be fairly constant 
in health, but show great variation, especially in infectious conditions. His 
first subdivision, known as Class i, is divided into (a) M cells, which are 
mononuclear forms identical with Ehrlich's myelocyte, (b) W cells, represent- 
ing forms which show only a slightly indented nucleus, the indentation never 
extending beyond the middle of the nucleus; this cell forms what is called the 
metamyelocyte, (c) T cells, in which the indentation of the nucleus is deeper 
than in the W cell, but there is no distinct separation into isolated loops, this 
form constituting the true polymorphonuclear type. The first two varieties of 
Class I are usually seen only under abnormal conditions, although the W cell 
may be found to the extent of 0.2 per cent, in healthy conditions. The cells 
of Class I are usually present, according to Arneth, to the extent of 5 per 
cent., Simon giving this percentage as from 4 to 9. The second class of 
Arneth embraces cells with two distinct nuclear fragmentations and shows 
three subdivisions: {a) 2 K cells, neutrophiles whose nucleus consists of two 
round nuclear portions; {b) 2 S cells, neutrophiles whose nucleus consists of 
two distinct S-shaped forms; (c) i K i S cells, neutrophiles whose nucleus 
consists of one round nuclear portion and one S-shaped division. These cells 
of Class 2 constitute, according to Arneth, 35 per cent, of the total neutro- 
philes, while Simon's figures are 21 to 47 per cent. In Arneth 's figures we 
find the 2 S cells forming about 23 per cent, of the total of this class. The 
third class has three nuclear divisions and is subdivided into four parts, as 
follows: (a) 3 K cells, (b) 3 S cells, (c) 2 K i S cells, (J) 2 S i K cells. Arneth 
gives the percentage of Class 3 as 41, the 2 K i S and the 2 S i K subdivisions, 
each representing approximately 16 per cent, of the total of this class. Si- 
mon's figures for this class range from 33 to 48 per cent. The fourth class 
comprises cells with four nuclear divisions showing five subgroups, as follows: 
(a) 4 K cells, (b) 4 S cells, (c) 3 K i S cells, (^) 3 S i K and (e) 2 K 2 S cells. 
This class, from Arneth 's figures, shows a percentage-of 17, the 4 K, 3 K i S, 
and 2 K 2 S types being largely in excess of the other forms. The fifth class 
comprises cells with five or more nuclear subdivisions and may be arranged 
into five groups, this class representing about 2 per cent, of the total neutro- 
philes. It is probable that these various classes represent the gradual develop- 
ment of the polymorphonuclear neutrophile, the older the cell the greater the 
tendency to reach Class 5, while in conditions associated with new formation 
of cells, as in infectious conditions, we' find the percentages of the earlier 

^ Jena, 1904. 
36 



562 DIAGNOSTIC METHODS 

classes being increased, that of the later ones diminished. Minor and Ringer^ 
have recently emphasized the prognostic value of this method in pulmonary 
tuberculosis. 

(b) Polymorphonuclear Eosinophiles. 

These cells, sometimes called the coarsely granular cells of Schultze, 
are somewhat smaller than the preceding, varying in size from that of a 
lymphocyte to that of the neutrophile. Their protoplasm is usually some- 
what less in amount than is that of the neutrophile and may not be distinct. 
It is filled with coarse, round, or slightly oval granules about one micron 
in diameter, which are very refractile and appear in the fresh specimen dis- 
tinctly black, while in the stained smear they take up the acid portion of the 
dye and are called, therefore, acidophilic, oxyphilic, or eosinophilic cells. ^ 
These granules are the a granules of Ehrlich, and are sometimes associated 
with the /? granules of Ehrlich which are about the same size or a little smaller 
than the a granules and take both the acid and the basic stains, although 
with the ordinary staining solutions they appear, like the a forms, stained 
with eosin. The nuclei of these cells are coarsely reticulated, are larger and 
thicker than those of the neutrophiles, are usually bilobed, and more fre- 
quently show distinct separation of these lobes. These nuclei do not "stain 
very deeply with the nuclear dyes, so that they may be rather indistinct. 
These cells are probably derived from the mononuclear eosinophile myelo- 
cytes of the bone-marrow and constitute from two to four per cent, of the 
total number of leucocytes, their average number being from 100 to 200 cells. 

(c) Polymorphonuclear Basophiles. 

These cells, frequently called mast cells, resemble the neutrophile cells 
in the fresh specimen, but show quite distinct characteristics in the stained 
form. Their protoplasm is much the same as that of the neutrophile and 
shows the same relation toward the nucleus which does not, however, so 
frequently form distinct lobes as does the nucleus of the neutrophile. The 
size of these cells averages about 10 microns, but shows a marked variation, 
being very small in myeloid leukemia. These cells are characterized by 
their granules, the 7 granulation of Ehrlich, which are more irregular in size 
than are the neutrophile granules and are not so extensively scattered through 
the protoplasm. These granules show the peculiar property of metachro- 
masia, being colored red with violet dyes and with the blue dyes violet, although 
with absolutely pure methylene blue they take a blue shading. In cases of 
myeloid leukemia these granules are particularly soluble in water and may, 
therefore, not be seen if aqueous stains are used, while in normal blood the 

^ Am. Jour. Med. Sc, 1911, CXLI, 638. See, also, Ringer, Ibid., 1912, CXLIV, 561; 
Kramer, New York Med. Jour., 1913, XCVII, 1241; Cummings, Calif. State Jour. Med., 
1913, XI, 286; Cooke, Brit. Jour. Tuberc, 1914, VIII, 211; Macfie, Ann. Trop. Med. and 
ParasitoL, 1915, IX, 435; Jour. Trop. Med: and Hyg., 1916, XIX, 41; Kahn, Jour. Lab. 
and Clin. Med., 1916, I, 599; Burgess, Ibid., 191 7, II, 240. 

2 See Miiller (Wien. klin. Wchnschr., 1913, XXVI, 1025) for a discussion of the chemical 
properties of these granules. Acton and Knowles (Indian Jour. Med. Research, 1914, I, 
523) believe the Kurloff bodies of the bone-marrow to be forerunners of the eosinophile 
granules. See, also, Pappenheim, Folia Haemat., 1914, XVIII, 224; Downey, Ibid., 1915, 
XIX, 148; Photakis, Ztschr. f. exp. Path. u. Therap., 19 15, XVII, 27c. 



PLATE XXI. 






* > 



»» . ^;^ 









♦ . «! 






r ^ 



lODOPHILlA. 



THE BLOOD 563 

granules are usually water-fast. These cells stain best with Ehrlich's dahlia 
stain, or Tiirk's iodin solution, and take a tone which is not strictly baso- 
philic, but resembles more that of mucin, on which account they have been 
called mucinophiles. Whether these granules are all of the same significance is 
questionable. The true mast cell granules are known as the 7 granules, while 
other basophilic granulations have been found by Ehrlich and are known as 
the delta (5) granules.^ These latter granules are found in the large mononu- 
clear cells, especially in the lymphocytes, and do not stain with Ehrlich's 
dahlia stain. Whether these latter bodies are true granules or nodal thick- 
enings is at present uncertain. True mast cells probably originate in bone- 
marrow from a granular mononuclear type corresponding to other types of 
myelocytes (Pappenheim). These cells constitute about 3^^ per cent, of the 
total leucocytes, averaging between o and 50 per cmm. 

Ehrlich's dahlia stain for these mast cells is made as follows: 

Distilled water, 100 c.c. 

Saturated absolute alcohol solution of dahlia, 50 c.c. 
Glacial acetic acid, 10 c.c. " 

The specimens are heated or fixed by alcohol and are then stained in the 
above stain for 5 to 10 minutes, the mast cells appearing of a distinctly 
violet tone. 

Turk's Iodin Method. 

The specimens are fixed with heat and are then first stained in a i per 
cent, alcoholic methylene blue solution, warming the slide very carefully until 
it steams. It is then allowed to cool, washed quickly in water, and dried 
between filter-paper. The slide is then covered with a solution of iodin in 
potassium iodid of the strength i : 300. Allow this solution to act not longer 
than }/2 minute, pour off and mount in the following syrup: 

Iodin, I gram. 

Potassium iodid, 3 grams. 

Distilled water, 100 grams. 

Gum arable q. s. to make a syrup. 

The mast-cell granules appear, when treated in this manner, very dis- 
tinctly outlined and colored black. The nuclei are brownish in color, the 
erythrocytes yellowish-green, and the polychromatophilic erythrocytes dark 
green, the neutrophile and eosinophile granules faintly yellow. 

(C) Leucocytes in Pathological Blood. 

(i) Myelocytes. 

Under this heading we must regard any cell of the bone-marrow as a 
myelocyte, but for diagnostic purposes we have reference more to mononuclear 
ceils which are distinctly granular. While the granulations of these cells are 

^ Huguenin (Zentralbl. f. allg. Path. u. path. Anat., 191 2, XXIII, 725) reports the 
presence of sudanophile granules in the mast cells in an obscure case. 



564 DIAGNOSTIC METHODS 

usually either neutrophilic or eosinophilic, we may rarely find, especially in 
myelogenous leukemia, cells which show basophile granulations. The size 
of these myelocytes varies from that of the red blood-corpuscle to that of a 
large mononuclear cell. 

(a) Neutrophile Myelocytes. 
This myelocyte may be either large or small, in the first case being known 
as Cornil's myelocyte (amblyochromatic type), a cell which is much larger 
than the polymorphonuclear leucocyte (at least 1 5 microns in diameter) , and 
showing a round, pale, eccentric nucleus which stains feebly but not diffusely. 
This cell shows many distinct neutrophile granulations and has a narrow zone 
of basophilic protoplasm surrounding the nucleus. It is found almost en- 
tirely in myelogenous leukemia and has been called by Pappenheim the 
heteroplastic promyelocyte. 'The second type of neutrophile myelocyte is 
known as the Ehrlich myelocyte (trachyochromatic type of Pappenheim) 
which is a medium-sized cell with a pale central nucleus which stains deeply 
but not diffusely. This cell shows extensive neutrophilic granulation of the 
protoplasm, which is faintly oxyphilic, and has a nucleus which is either per- 
fectly round, oval, or indented, but is never lobed nor pycnotic. A distinc- 
tion between the myelocyte and the polymorphonuclear leucocyte should be 
based entirely upon the structure of the nucleus, all those cells with round, 
oval, or kidney-shaped nuclei which occupy at least one-half of the cell and 
show neutrophile granulations but no diffuse staining of the nucleus must be 
called myelocytes. It is to be remembered, however, that this type develops 
into the polymorphonuclear neutrophile, so that in abnormal blood we may 
have all gradations between these two types. 

(b) Eosinophile Myelocytes. 
These cells are exactly analogous to the preceding, with the exception that 
the granules of the more mature form show distinct eosinophilic tendencies. 
The younger forms of these granules may show a purplish- violet or even blue 
color, owing, as Simon states, to the fact that the young eosinophilic granule is 
physically cyanophilic and chemically amphophilic, whereas the mature 
granule is physically erythrophilic, but chemically absolutely oxyphilic. 
The size of these cells is more or less variable, so that it is probable that we 
have the two types of eosinophile myelocytes, corresponding to the large and 
small neutrophile myelocytes. These cells occur more frequently in leuke- 
mia, in association with tumors of the bone-marrow^ and in the pseudoleu- 
kemic anemia of children. 

(c) Basophile Myelocytes. 

These myelocytes may be of variable size, but are characterized by the 

large centrally located nucleus, which is not clearly defined from that of the 

surrounding slightly basophilic protoplasm. The granules are distinctly 

basophilic and in some cases are very numerous, while in others they may be 

widely scattered through the protoplasm. ^ These cells are practically never 

^ von Roznowski (Ztschr. f. klin. Med., 1915, LXXXI, 377) believes that the presence 
of myelocytes in large numbers in cases of cachexia points to metastasis in the bone-marrow. 
- See Aubertin and Chabanier, Ann. de Med., 1915, II, 399 



THE BLOOD 565 

seen except in cases of severe splenomyelogenous leukemia, in which they may 
reach as high as 47 per cent, with an absolute count of 140,000 cells (Taylor). 

(2) Irritation Forms. 

These cells vary in size from a lymphocyte to a large mononuclear cell, 
resembling more nearly the former. They are mononuclear, nongranular 
cells, thus differing from the myelocyte which is always granular. The 
nucleus is round and eccentrically placed, showing a very slight chromatin 
network and staining with the triple stain of a bluish-green color 
while with the Romanowsky dyes the color is a pale blue. The protoplasm 
is stained a deep brown with the triple stain and is thus differentiated 
from other forms of cells. With the methylene blue dyes the protoplasm 
appears more deeply stained than does the nucleus. These cells were first de- 
scribed by Tlirk and would seem to have the same significance as do the myelo- 
cytes, namely, an indication of marked activity of the bone-marrow. Pappen- 
heim regards them as plasma cells and largely derived from the lymphoctyes. 

(3) Degenerated Forms. 

Occasionally we find in normal blood degenerated leucocytes which stain 
poorly and show no granules. These may be even so much degenerated that 
they show as the so-called basket cells or "shadows." This condition is 
very frequently seen in severe infectious diseases, while in the acute leucocy- 
toses which occur under many influences diminution in the number of neutro- 
phihc granules as well as swelling and fragmentation of the bodies of the 
leucocytes is very common. The changes in the staining qualities of the 
nucleus seem to be the most significant of the lesions in the acute type of 
degeneration of the leucocytes. In chronic degeneration of the leucocytes we 
find hydropic degeneration, which is frequent in the blood of chlorosis and, 
when the nuclei are involved in this degeneration, seems to be limited to 
certain cases of leukemia. Besides such changes we find fatty degeneration 
as well as glycogenic degeneration. This fatty degeneration is characterized 
by the appearance of fat globules in the leucocytes which stain with osmic 
acid and with Sudan-Ill. For this latter reason they have been styled 
sudanophiles, and have been very carefully studied by Buttini and Comesatti, 
as well as by Cesaris-Demel. 

lodophilia. 

In many pathologic conditions, especially in acute infectious diseases 
and in those associated with all types of sepsis, a so-called glycogen reaction or 
iodophilia may be demonstrated in the bodies of the leucocytes, as well as in 
certain extracellular granules. The technic of this method is as follows: 
An unfixed dry blood smear is exposed to the vapor of solid iodin until it is 
stained a brownish color. After the specimen is stained it is mounted in the 
syrup described on page 563 and is examined with an oil-immersion lens. 
The blood of normal individuals, stained by this method, shows the proto- 
plasm of the leucocytes of a bright yellow, while the nucleus takes on a much 
lighter tint. In pathologic blood two types of reaction can be noted. The 
intracellular one, which is of greater clinical importance, shows a more or less 



566 DIAGNOSTIC METHODS 

marked diffuse brown color of the entire protoplasm of the leucocyte, or the 
protoplasm contains reddish-brown granules which may be more or less dis- 
tinct. The extracellular reaction is evident in the blood plates, while the 
intracellular type is more particularly confined to the neutrophiles, although 
the mononuclear leucocytes may occasionally be tinged brown. Much 
difference of opinion exists as to the nature of this brown-staining substance, 
Ehrlich regarding it as glycogen, while Czerny considers it as an antecedent 
of amyloid, and Goldberger and Weiss regard it as peptone. Kaminer regards 
this reaction as a degenerative change and not as an evidence of regeneration. 
While this reaction has little value in differentiating infectious or septic condi- 
tions one from the other, it is sometimes of importance in making a diagnosis 
between purulent and nonpurulent affections, being present in the former 
and absent in the latter. These granules have been found by Hofbauer in 
pernicious anemia, secondary anemia, and leukemia, but not in chlorosis or 
pseudoleukemia. This reaction is observed in pneumonia, but is seldom seen 
in tuberculosis, typhoid fever and diphtheria. It is, however, not to be re- 
garded as dependent upon infection, as it occasionally obtains in non-infec- 
tious conditions. 

(D) Differential Counting of the Leucocytes. 

By a differential counting of the leucocytes is meant the counting of the 
different varieties of the leucocytes found in the stained smear and the calcula- 
tion of each type in terms of percentage. The technic of this method is 
that previously outUned and consists in making an even smear upon a glass 
slide and staining it with any of the stains previously mentioned, noting that 
the triple stain does not bring out the granulations of the leucocytes with the 
exception of those of the neutrophiles.^ It is self-evident that the larger 
the number of leucocytes counted the greater will be the possibility of arriv- 
ing at true percentage relations. It is wise, therefore, to count at least 250 of 
these cells, and in many cases, to extend this to 500. If the smear is even 
and the leucocytes well distributed throughout, 100 cells will frequently suffice. 

For a differential count a satisfactory classification is an absolute essen- 
tial. As none of the systems at present advanced are entirely adequate, we 
still use the classification of Ehrlich, which is as follows: SmaU mononuclears, 
large mononuclears (including the transitional), polymorphonuclear neutro- 
philes, eosinophiles, basophiles (mast cells), and myelocytes. The character- 
istics of these cells have been previously given, but may be summed up in this 
connection. By a small mononuclear is meant any nongranular mononu- 
cleated cell smaller than a polymorphonuclear neutrophile. A large mononu- 
clear is any nongranular cell with a round or oval nucleus and larger than 
a polymorphonuclear neutrophile. A cell of the same description and size 
but with an indented nucleus is a transitional form. The polymorphonuclears 
are cells which are about 10 microns in size and show a diffuse granulation, 
which may be either neutrophile, eosinophile, or basophile. It should be 
remembered that the mast cells appear as nongranular forms when the triple 

^ See Arneth, Deutsch. Med. Wchnschr., 1913, XXXIX, 2560; also, Dunzelt, Miinch. 
Med. Wchnschr., 1913, LX, 2616. 



THE BLOOD 567 

stain is used, so that the characteristics of the nucleus in its relation to the 
protoplasm must be borne in mind. The percentage relations of these cells 
in normal blood are as follows:^ 



Percentage. 


Number per cmm. 


20-25 


1200-2000 


3-5 


200-400 


65-75 


5000 


2-4 


100-200 


<y~V2 


0-50 



Small mononuclears, 
Large mononuclears, 
Polymorphonuclear neutrophiles. 
Polymorphonuclear eosinophiles, 
Polymorphonuclear basophiles. 

In the writer's laboratory differential staining is usually carried out with 
the use of the Wright or Giemsa stain as he has found the usual triple stain 
quite unreliable. Although the granular differentiation is not as distinct as 
could be desired, yet one soon becomes accustomed to the staining of various 
cells so that it is not a matter of great difficulty to distinguish the various 
types. In many cases it is not the easiest matter to distinguish myelocytes 
from the small mononuclear types, but careful study will usually clear up any 
obscurities which exist. 

(£) Number of Leucocytes. 

The normal number of leucocytes in a cmm. of blood has beeri given vari- 
ous figures. As a rule, it may be said that anything above 10,000 leucocytes 
per cmm. should be considered pathological, the normal variation running 
from 5,000 to 9,000 cells. In estimating the normal number of white cells, 
both in health and in disease, a large number of factors which influence these 
cells must be taken into consideration. Thus vasomotor phenomena, varia- 
tions in the volume of the plasma, inflammatory processes, state of digestion, 
age, variations in different parts of the circulatory system, and many different 
disease processes usually bring about an increase in this number, while many 
pathological conditions are associated with a reduction. An increase in the 
number of white cells is usually spoken of as a leucocytosis, but it must be 
remembered that such an increase may be purely physiological and should be 
sharply differentiated from a pathological increase which is clinically the more 
important. This increase is usually referable to the increase in the number of 
the polymorphonuclear neutrophiles, while an increase in the other varieties 
of cells is spoken of as a lymphocytosis, myelocytosis, esoinophilia or an 
eosinophilocytosis, or a mixed leucocytosis. 

A diminution of the number of polynuclear neutrophiles is designated as a 
leucopenia, which is the more usual form in which reduction of the cells 

^ See Bunting, Am. Jour. Med. Sc, 1911, CXLII, 698; Galambos, Folia Haemat., 1912, 
XIII, 153; von Torday, Virchow's Arch. f. path. Anat., 1913, CCXIII, 529; Mehrtens 
(Arch. Int. Med., 19*3, XII, 198) calls attention to the frequency of relatively low poly- 
nuclear leucocyte counts with high lymphocyte counts in the routine clinical work. Warfield 
(Jour. Am. Med. Assn., 1915, LXIV, 1296) advises subdivisions of these groups so that we 
have seven classes. Leo-Wolf, Interstate Med. Jour., 191 5, XXII, 1235; Gruner, Brit. 
Jour. Surg., 1916, III, 506; De Boe, Florida Mei. Assoc. Jour., 1916, II, 294; Turban, Ztschr, 
f. Turberk., 1917, XXVI, 242. Cardenal, (Sig. Med., 1916, LXIII, 785) confirms the value 
of Sondem's line of resistance as applied to the differential count. See, also. Jour. A. M. 
A., 1917, LXVIII, 730; Blumberg, Am. Jour. Syph., 1918, II, 734; Mcjunkin and Charlton,. 
Arch. Int. Med., 1918, XXII, 157. 



568 DIAGNOSTIC METHODS 

occurs. It is to be remembered that these conditions may be transitory and 
symptomatic pointing to a purely physiological process, while a more per- 
manent and more marked increase or decrease in the number should be con- 
sidered pathological. It is rare that we find the increase limited absolutely 
"to one variety of cell, the increase in the others being less marked. The 
absolute number of these cells is much more to be regarded than their per- 
centage relation, as with an increased number of leucocytes the actual number 
of some of these varieties may be increased, although the percentage may be 
diminished; while with a low leucocyte count the percentage may be increased 
and the absolute number diminished. It is wise, therefore, to report not 
only the percentage relations of the leucocytes, but also the actual nimaber of 
these cells per cmm. This will correct mistaken ideas as to an apparent in- 
crease or decrease in any particular variety of cell. Thus a differential 
leucocyte count may show a percentage of 50 for the neutrophiles and at the 
same time an actual number of 10,000 per cmm., giving rise to confusion as to 
the actual relation of these important leucocytes. 

Leucocytosis (Polymorphonuclear Neutrophilosis). 

As stated above, anything above 10,000 leucocytes per cmm. should be 
regarded as a leucocytosis. This is, however, relative and should not be con- 
sidered pathological without taking into consideration all of the physiological 
and pathological influences. Regarding the theoretical cause of leucocytosis, 
little is definitely known. The influence of infectious processes is such as 
usually to increase the number of leucocytes in the blood, as an attempt on 
the part of the system to overcome by phagocytosis the action of the bacteria 
of the various diseases. Yet we find in some of tliese infectious processes, 
notably in typhoid fever, a marked reduction in the number of the white cells, 
although the bacillus typhosus is present in large numbers in the blood at the 
same time. • We must, therefore, assume some specific influence upon phago- 
cytosis and chemotaxis, as a general infection is not necessarily associated 
with an increase in the number of white cells, but is dependent more upon 
the specific nature of the infection. The work upon opsonins and vaccine 
therapy may open up an entirely new field in our study of this subject. 
The leucocytosis shown in noninfectious conditions is still a matter of much 
dispute and has probably more to do with variations in the plasma than with 
direct increase in the number of white cells. 

Classification. 

According to Limbeck,^ the following classification of the various leuco- 
cytoses is the most comprehensive: 

(i) Physiological leucocytosis. 

(a) Leucocytosis of digestion. 

(b) Leucocytosis of pregnancy. 

(c) Leucocytosis of the new-born. 
(2) Pathological leucocytosis. 

(a) Inflammatory leucocytosis. 
'■ Jena, 1896. 



PLATE XXII. 




POLYNUCLEAR LeUCOCYTOSIS. (WrIGHT's StAIN.) 



THE BLOOD 569 

(b) Leucocytosis associated with malignant tumors (cachectic 
leucocy tosis) . 

(c) Posthemorrhagic leucocytosis. 

(d) Agonal or antemortem leucocytosis. 

(3) Leucocytosis following medicinal and therapeutic measures. 

(4) Leucocytosis from other causes. 

(i) Physiological Leucocytosis. 
(a) Leucocytosis of Digestion. 

Normally a leucocytosis of the polynuclear type will be observed beginning 
about one hour after a meal rich in proteins and will reach its maximum in 
from three to five hours. ^ The actual figure reached varies in different per- 
sons, being usually an increase of about one-third, the maximum often reach- 
ing 15,000 cells, but more usually not much over 10,000. In this increase the 
small mononuclear cells may be absolutely as well as relatively increased. 
In some persons this leucocytosis of digestion does not appear, which fact 
may be referable to marked torpidity of the intestines, to a prolongation of 
the process of digestion, or to a large absorption of fluids. It has been found 
that a highly albuminous diet has a much more marked influence upon this 
leucocytosis than does a diet of vegetables and fat. The rapidity of ab- 
sorption and of digestion must be taken into consideration as factors which 
influence the appearance or nonappearance of the leucocytosis. In children 
the increase in cells is much more marked than in the adult, due probably to 
the increased digestive and absorptive powers, providing the food taken is, as 
it should be, easily digestible and absorbable. 

Various pathologic conditions influence this digestion leucocytosis. Thus 
Miiller has found that in cases of carcinoma of the stomach a digestion 
leucocytosis is rarely observed after heavy meals. There are, however, a few 
cases of gastric cancer in which a slight increase has been observed. This 
failure of leucocytosis is probably due, as Schneyer shows, to lessened ab- 
sorption as a result of involvement of the lymphatics rather than to a malig- 
nant stenosis of the pylorus, as the benign stenoses are usually associated with 
a leucocytosis. That the lack of leucocytosis is due more to diminished ab- 
sorption than to lack of digestive power is shown by the usual occurrence of a 
digestion leucocytosis in ulcer of the stomach, chronic gastritis, and in dyspep- 
tic conditions. Just why carcinoma, with or without stenosis, should be 
associated with a normal or subnormal number of leucocytes after digestion is 
hard to explain, in view of the fact that these other conditions are associated 
with a leucocytosis. Practically all absorption of food material occurs from 
the bowel and we would naturally expect to find disorders of the intestinal 
canal leading more frequently to a normal leucocyte count than to a leucocyto- 
sis. Very little detailed work has been done upon the influence of enteric 
troubles upon the leucocyte count, but the writer has seen several cases in 
which a digestion leucocytosis did not occur and in which the findings both 
ante- and postmortem were entirely related to the bowels. The examination 

^ See Brasch, Ztschr. f. exp. Path. u. Therap., 191 2, X, 381; also, Mitchell, Am. Jour. 
Dis. Child., 1915, IX, 358. 



570 DIAGNOSTIC METHODS 

of the blood may, however, clear up a diagnosis of carcinoma, but will not 
always permit of a differential diagnosis between this condition and pernicious 
anemia. It is to be remembered that a leucocyte count must be frequently 
made in order that this test may be of any value whatever and that a consider- 
able rise only is to be taken as evidence of a leucocytosis. Patients with 
cancer either of the stomach or of other viscera frequently show a leucocytosis 
as the result of cachexia and this should be remembered in the interpretation 
of a leucocyte count following digestion. 

(b) Leucocytosis of Pregnancy. 

It has been shown that from 50 to 75 per cent, of cases of pregnancy are 
associated with a leucocytosis, averaging about 15,000 cells per cmm. This 
is especially true of primiparae, but is often shown in the multiparas. Just 
what cause can be given for this rise is uncertain. It is more than likely that 
a condition of slight intoxication is present due to the overloading of the 
blood of the patient with the products of metabolism of the fetus. These sub- 
stances are not normal to the blood of the woman and in consequence act 
as foreign bodies which may attract, by their chemotactic influence, the white 
cells and bring about an increase. As these cases of pregnancy show an ab- 
sence of a digestion leucocytosis it has been assumed that the increase in 
leucocytes is due to a prolonged digestion leucocytosis, but this does not seem 
probable. The changes in the breasts and in the uterus during this period 
have suggested these organs as the factors influencing this leucocytosis, but 
such inferences do not seem to be directly warrantable. The leucocytosis of 
pregnancy is a mixed leucocytosis, all of the various types of leucocytes, with 
the exception of the eosinophiles being increased. After the birth of the child 
the leucocytes gradually diminish in number and normally reach their usual 
values in from four to fourteen days after delivery. Should complications, 
such as postpartum hemorrhage or septic fever arise, the leucocyt:e count may 
remain high until these complications have subsided. In multiparae these 
changes in the number of leucocytes are not so marked as in the case of the 
primiparae but a slight rise always occurs. This fact has been attributed to 
the lessened reactivity of the organisms to the influence of the toxic substances 
thrown into the blood from the cells of. the fetus. ^ 

(c) Leucocytosis of the New-bom. 

As Askanazy has found, the blood of the fetus shows a diminution in the 
number of leucocytes, owing to the fact that there is no function as yet estab- 
lished for these cells in utero. In the blood of the new-born, however, a 
leucocytosis running from 15,000 to 20,000 cells may be observed, these fig- 
ures going as high as 40,000 under the influence of the first feeding. As the 
weight of the child begins to diminish these cells are markedly reduced in 
number to about 8,000, and are subsequently increased to 10,000 as the child 
begins to gain in weight. The high leucocyte count at birth is more probably 
due to the rapid blood formation than to a concentration of the blood, al- 
ij though this latter factor as well as that of the influence of digestion must be 

1 See Baer, Surg, Gyn. and Obs., 1916, XXIII, 567. 



THE BLOOD 571 

taken into consideration. The increase in the number of cells is chiefly limited 
to that of the small mononuclear cell, the differential count of the leucocytes 
showing during the early periods of life from 40 to 60 per cent, of the total 
number. This leucocyte count, if taken at the moment of birth, will not, 
however, vary much from that of the adult, the change becoming more 
marked in the early periods of life. 

(2) Pathological Leucocytosis. 
(a) Tnflammatory Leucocytosis. 

In most cases of inflammatory nature, of acute infections and general 
febrile diseases, there is observed an absolute increase of the polymorphonu- 
clear neutrophiles, which runs more or less parallel to the temperature. This 
increase may run from 10,000 to 50,000 cells and diminishes as the influence 
of the inflammatory process is diminished. In general it may be said that 
leucocytosis represents the reaction of the individual to the disease. A high 
count may mean a vigorous reaction to the infection; a low count may 
mean either a poor reaction and hence an unfavorable condition of 
the patient, or it may indicate a very mild degree of infection with a normal 
reactivity of the patient. It must be said that all diseases of an infectious 
nature are not necessarily associated with a leucocytosis. For instance, 
pneumonia shows a leucocytosis which runs parallel to the degree of viru- 
lence; measles, influenza, malaria, and tuberculosis are rarely if ever associ- 
ated with a leucocytosis unless complications arise or the conditions become 
very severe.^ In typhoid fever we usually find a leucopenia which, howeveir, 
is associated with a relative lymphocytosis; if complications such as perfora- 
tion arise a leucocytosis may appear. Among the conditions causing leucocy- 
tosis are acute lobar pneumonia, the count running between 20,000 and 
100,000, depending upon the severity of the infection and the degree of resist- 
ance against the infection. Acute articular rheumatism, diphtheria, acute 
cerebrospinal meningitis, follicular and suppurative tonsillitis, scarlet fever, 
mumps, rabies, erysipelas, ulcerative endocarditis, small-pox, cholera, general 
pus infections of the serous membranes and of the mucous membranes,^ acute 
bronchitis, and many other conditions cause a leucocytosis of the polymor- 
phonuclear type. The variations in these many conditions will be discussed 
under the Pathology of the Blood. 

The exact cause of this leucocytosis following inflammatory processes 
is still a matter of much discussion. The bone-marrow has been shown to be 
markedly increased as regards cellular proliferation in the early stages of the 
inflammation and may be so markedly changed as to cause permanent de- 
rangement in the functions of this blood-forming organ. That leucocytosis 
has much to do with immunity both from the standpoint of phagocytosis and 
from that of Ehrlich's side-chain theory cannot be questioned.^ A leucocy- 

^ See Hess, Am. Jour, Dis. Child., 1914, VII, i; McDougall, Brit. Jour. Tuberc, 191 7, 
X, 159; Govaerts, Presse Med., 191 7, XXV, 180; Forbes and Snyder, Jour. Lab. and Clin. 
Med., 1918, III, 758; Friedman, Am. Jour. Med. Sc, 1919, CLVIII, 545; Douglas, Bull. 
Johns Hopk. Hosp., 1919, XXX, 338. 

2 See Smith, Surg. Gynec. and Obst., 1913, XVI, 403. 

3 Gay and Claypole (Arch. Int. Med., 1914, XIV, 662) call attention to the specific 
hyperleucocytosis following inoculation with typhoid vaccines. 



572 DIAGNOSTIC METHODS 

tosis must represent the attempt on the part of nature to rid the blood and 
the system of the bacterial and toxic products of the disease. Whether 
phagocytosis as such or under the influence of various opsonins is the direct 
cause of immunity and of recovery from any specific infection must be left to 
a later chapter. 

(b) Cachectic Leucocytosis. 

This variety of leucocytosis is the least uncertain. The cases of carci- 
noma and of sarcoma show a leucocytosis which is not definite for the particu- 
lar kind of cancer, but is more usual with the latter than with the former 
type. The leucocytosis is usually one of the polymorphonuclear type, but 
is frequently associated with an increase in the number of mononuclear 
cells. This leucocytosis has no direct relation with the situation of the tumor 
and is not always present in all cases of cancer. Whether this leucocytosis 
be due to an intercurrent infection is a question which must be left for more 
detailed work, but it would seem wise to accept Ewing's statement that a 
marked leucocytosis in the course of a cachexia, from tertiary syphiHs, tuber- 
culosis, nephritis, and in the majority of cases of carcinoma, should suggest 
a search for a complicating infection, while in the sarcomata a leucocytosis is 
much more common as a direct result of the disease. 

(c) Posthemorrhagic Leucocytosis. 

A well-marked leucocytosis, which may begin in from ten to fifteen minutes 
and may reach as high as 20,000 cells within an hour, has been of ten observed 
following extensive acute hemorrhages. The leucocytosis in these cases bears 
a general relation to the extent and rapidity of the loss of blood and usually 
disappears or diminishes long before regeneration of the blood has occurred.^ 
This leucocytosis is of the polymorphonuclear type and is referable rather to 
the sudden outflow of lymph which occurs as a compensation for the loss of 
fluid than to a new production of cells, as this latter process does not take 
place for some time. In hemorrhages which are slight and long-continued 
as in cases of gastric or intestinal ulcer, the duration of the leucocytosis as 
well as its extent is very brief. Stassano and Billou assume from their work 
that a hypoleucocytosis follows a severe hemorrhage while a trueieucocytosis 
is observed, after the loss of small qauntities of blood. These findings seem to 
be rather doubtful, except where the hemorrhage has been so extensive as to 
cause death. 

{d) Antemortem Leucocytosis. 
This form of leucocytosis has been questioned, especially by Arneth, but 
there seems to be little doubt that such a leucocytosis may occur if death does 
not take place too rapidly. In some diseases the leucocytes do not fall in 
number, but in some a distinct rise is noted which has been attributed by 
Ehrlich to the accumulation of white cells along the periphery of the blood- 
vessels as a result of slowing or stasis of the circulation. This type of leuco- 
cytosis is usually of the polymorphonuclear variety and may support the view 

^ SeeLevison, Jour. Am. Med. Assn., 1915, LXIV, 1294; Dold, Mitt. a. d. Grenzgeb. d. 
Med. u. Chir., 1916, XXIX, 68. 



THE BLOOD 573 

of Limbeck that antemortem leucocytosis, when it does occur, is the result of 
a terminal infection, although it cannot explain those cases which show a 
lymphocytosis rather than the ordinary leucocytosis. The character of the 
antemortem leucocytosis must depend largely upon the precedent condition 
and will be associated with antemortem dissemination of bacteria, antemor- 
tem hyperpyrexia, vasomotor paralysis, serous exudation, etc. 
(3) Leucocytosis Following Therapeutic Measures. 

(a) Drugs. 
It has been found following administration of tonic drugs, ethereal oils, 
myrrh, turpentine, camphor, peppermint, quinin, and other drugs, that a 
leucocytosis of a more or less extent occurs, which is probably referable to the 
same cause as is digestion leucocytosis. Many of these drugs if applied 
locally for the purpose of counterirritation were shown to have the same 
effect. Extracts of tissues, especially those containing large amounts of 
nucleinic acid and of purin substances, produce an extensive leucocytosis, 
which fact has been taken advantage of in the administration of the nuclein 
substances as therapeutic remedies. In the case of those drugs which de- 
stroy the red cells, such as the coal-tar antipyretics, chlorates, and illuminat- 
ing gas, a normal number of white cells is usually observed, although Simon 
wrongly states that a hyperleucocytosis follows the use of such drugs. After 
the prolonged use of chloroform or ether a polynuclear leucocytosis is gener- 
ally observed which is usually of short duration.^ In this connection it is well 
to remember that an increase in the number of leucocytes of 10,000 to 15,000 
above the normal value of the individual should be regarded as evidence of 
infection if this increase is sustained for more than a few hours. 

(^>) Baths. 
After a cold bath the leucocytes of the polynuclear variety have been 
shown to be increased from 100 to 300 per cent.^ This is true only if the bath 
is of moderate duration, a prolonged cold bath taken to the point of exhaus- 
tion will diminish rather than raise the number of white cells. Experiments 
on the result of hot baths have shown just the reverse condition, namely, a 
hot bath of short duration produces a decrease, while one of long duration 
causes an increase in the number of white cells. Massage in itself following 
either a hot or cold bath tends to increase the number of leucocytes. 

(c) Exercise. 
Prolonged muscular exercise, as taken in the form of gymnasium work, 
as applied in the various therapeutic movement treatments, and also as given 
in the more violent athletic contests produces a rise in the number of leuco- 
cytes which is temporary and is characterized more by an increase in the num- 
ber of polynuclear cells than of any other variety of the leucocytes. 

(4) Leucocytosis from Other Causes. 

Cyanosis, passive hyperemia obtained by the method of Bier, shock 

whether physical or mental, injection of various toxins, such as Koch's tuber- 

^ See Mann, Jour. A. M. A., 1916, LXVII, 172. 

2 See Rovighi and Secchi (Rif. Med., 1914, XXX, 617; Miinch. Med. Wchnschr./i9T4, 
LXI, 1 721) for a discussion of leucocytosis following exposure to cold. 



574 DIAGNOSTIC METHODS 

culin, the various autovaccines, and injection of various organic principles, 
such as peptone, pus, organ extracts, etc., have all been shown to produce a 
polynuclear hyperleucocytosis, either by causing stasis of the circulation or by 
increasing the chemotactic power of the serum. ^ The exact explanation of 
chemotaxis is still in doubt and we find that some substances are positively 
and others negatively chemotactic, thus accounting for the lack of phagocy- 
tosis in certain conditions, such as pneumonia, although in these same condi- 
tions we may have a leucocytosis as a direct result of an infection. From the 
work of Rosenow we learn that the phagocytic action of the leucocytes in 
pneumonia is increased by the addition of an extract of the active pneumo- 
cocci; whether this extract increases the chemotactic power of the bacteria 
in the blood or whether certain restraining influences are overcome is still 
unsettled. Future work upon phagocytosis and upon the factors governing 
increased opsonic power in the various infections may show us what causes 
are effective in such conditions. 

In the previous discussion of leucocytosis we have had reference more 
to an increase in the number of polynuclear cells, this type being more prop- 
erly called polynuclear hyperleucocytosis. It is very rare that this condition 
exists in the absolutely pure state. 
Mixed Leucocytosis. 

By this is meant an increase of both granular and of nongranular cells 
of various types, its more common interpretation, however, being a leucocy- 
tosis characterized by an increase in the number of neutrophile myelocytes. 
This neutrophilic myelocytosis is best seen in leukemia in which the absolute 
number of myelocytes may reach 150,000, the increase in no other condition, 
according to Ehrlich, rising above 1,000 cells. The increase in the myelo- 
cytes in leukemia is not limited to those of the neutrophile variety, but is com- 
monly associated with a marked increase in the number of eosinophile 
types as well as with an increase in the number of mast cells. In pernicious 
anemia we may also find a large increase in the number of these myelo- 
cytes. These cells when increased in number are clinically significant of ex- 
haustion of the bone-marrow, but should never be interpreted in this way 
unless they remain while the leucocyte count is falling; in other words, they 
are significant only when constituting a large percentage of the leucocytes. 
Lymphocjrtosis. 

A relative or absolute increase in the number of lymphocytes is of fre- 
quent occurrence and has occasionally some significance. In judging of this 
condition the actual number of cells present as well as their numerical rela- 
tions to the other varieties of leucocytes must be considered. The term 
lymphocytosis must always be appHed to an absolute increase in the number 
of these cells, the normal number being between 1,200 and 1,500 per cmm. 
Physiologically we find this condition in infants and during a digestive leucocy.- 
tosis in the adult. ^ The child shows at birth from 50 to 65 per cent, of these 

^ See Veraguth and Seyderheim (Miinch. med. Wchnschr., 1914, LXI, 301) for the 
leucocytosis following use of electricity. 

2 Kaufmann, Jour. Am. Med. Assn., 1914, LXIII, 1104; Staines, James and Rosenberg 
(Arch. Int. Med., 1914, XIV, 376) report a lymphocytosis due to increased altitude. 



THE BLOOD 575 

cells which percentage gradually diminishes, reaching the normal percentage 
(20-25) about the age of puberty. 

Pathologically this condition is observed in poorly nourished children,^ 
those showing the "constitutio lymphatica," rachitis, whooping-cough, 
gastro-intestinal disturbances in the child, cervical adenitis, splenic tumors, 
in most infectious diseases of children, ^ and especially in lymphatic leukemia, 
in which as high as 90 per cent, of a largely increased leucocyte count is ref- 
erable to the small lymphocytes. In the splenomyelogenous form of leuke- 
mia and following removal of the spleen we observe a steady increase in the 
number of lymphocytes continuing during the first year. It must be remem- 
bered that a leucocyte count may be low and yet a lymphocytosis exist. 
This is shown in typhoid fever, amebic dysentery, chlorosis, pernicious anemia, 
scurvy, and other conditions in which the leucocyte count is low, the granular 
cells being diminished in number, but the lymphocytes being increased.^ 
In the congenital and also secondary acquired syphilis we find alymphocytosis 
which must be referred to involvement of the lymph-glands. The worker 
must not be led into a mistaken interpretation of his blood-findings, as en- 
larged lymph-glands may give the same picture of lymphocytosis as is seen 
in Hodgkin's disease, chronic and acute lymphatic leukemia. 
Eosinophilia. 
By this is meant an absolute increase in the number of eosinophilic cells. 
The average number of these cells is between one and two hundred per cmm., 
hence the term eosinophilia should be limited to those cases showing counts 
something above 250. We find a physiological eosinophilia during child- 
hood, the average increase being about i to 2 per cent, above the normal 
adult finding. No physiological relations have been established between eosin- 
ophilia and sex, pregnancy, menstruation, digestion, or old age; however racial 
distinctions are sometimes shown by eosinophilia, the natives of southern 
China showing between 15 and 20 per cent, of the leucocytes as eosinophiles. 
Pathological Eosinophilia. 

We observe variations in the number of eosinophiles in various affections 
of the bone-marrow. Thus in splenomyelogenous leukemia, we may find 
these cells increased as high as 30,000 per cmm. According to Ehrlich, a 
diagnosis of this form of leukemia is warranted only when we have an increase 
in the number of eosinophiles, but it cannot be doubted that cases are rela- 
tively frequent in which these cells are not increased. In sarcoma of the 
bone-marrow as well as in osteomyelitis and osteomalacia, we find these cells 
usually increased in number. In chlorotic conditions we may find the eosino- 
philes moderately increased, while in secondary anemias especially those 

1 See von Hoesslin, Munch, med. Wchnschr., 1913, LX, 11 29 and 1206. 

* See Marchand, Deutsch. Arch. f. klin. Med., 1913, CX, 359; Marcovici, Folia Haem., 
1915, XX, 136. 

3 Borchardt (Deutsch. Arch. f. klin. Med., 191 2, CVI, 182) shows that a lymphocytosis 
obtains in a large majority of cases of disease of the thyroid, hypophysis and suprarenal 
gland. This is oftentimes associated with a leucopenia. Cabot (Am. Jour. Med. Sc, 1913, 
CXLV, 335) calls attention to the occasional occurrence of a lymphocytosis in infections 
usually associated with a polynucleosis. See, also, Huhle, Deutsch. Arch. f. klin. Med., 
1914, CXIII, 455; Mayer and Gourdy, Sem. Med., 1916, XXIII, 475, 513; Ibid., 1917, 
XXIV, 39 and 113; Bristol, Jour. A. M. A., 1919, LXXII, 1048. 



576 DIAGNOSTIC METHODS 

following infection with parasites a marked eosinophilia may be observed.^ 
After extirpation of the spleen as well as in cases of chronic splenic tumor we 
find an eosinophilia which may run from lo to 40 per cent, higher than the 
normal, constituting as high as 40 per cent, of the total number of leucocytes.^ 
Whether or not a pure disease of the lymph-glands is associated with the 
eosinophilia is unsettled, but if metastases have extended to the bone, an 
eosinophilia of marked degree is usual. 

True bronchial asthma is always associated with an increase in the num- 
ber of eosinophiles, one case of Billings showing 54 per cent, of the total 
leucocytes as eosinophiles. This relationship in asthma is of importance 
from a diagnostic standpoint as asthmatic attacks from other causes are not 
associated with an eosinophilia. It is true that in emphysema a marked 
eosinophilia does occur so that we may find, in cases in which this condition 
complicates a tuberculosis, that an eosinophilia obtains. Tuberculosis of 
the lungs or of other tissues does not show an eosinophilia unless complicated 
by emphysema, cachexia, or secondary infection.^ 

A large number of skin diseases are associated with eosinophilia, the high- 
est count being reported by Zappert in a case of pemphigus showing 4,806 
cells per cmm. The occurrence of this condition in skin diseases depends 
not so much upon the nature of the lesion as upon its extent, intensity and 
lack of healing tendency. Many skin lesions are known to be produced by 
toxic agents which have special chemotactic influences over the eosinophile 
cells; this fact may account for the eosinophilia in such conditions. The 
most usual skin diseases showing this increase are pemphigus, eczema, 
psoriasis, urticaria, purpura, scleroderma, lupus, leprosy, herpes zoster, 
and general gouty affections.^ 

It is a general rule that the infectious fevers are not associated with eosino- 
philia, but with the acute polynuclear leucocytosis.^ In scarlet fever, how- 
ever, we find that eosinophiles are frequently markedly increased, .while in 
acute rheumatism and in malaria these cells are usually present in more or 
less increased numbers.^ In gonorrheal infections the eosinophile leucocytes 

^Beifeld and Barnes, Bull. Johns Hopk. Hosp., 1916, XXVII, 181; Grosso, Rif. Med., 
1916, XXXII, 314 and 340. 

2 See Giffin, Am. Jour. Med. Sc, 1919, CLVIII, 618. 

3 See Arneth (Deutsch. Arch. f. klin. Med., 1912, CVIII, 323), for the relations of the 
eosinophiles in pneumonia. Chuprin (Med. Obozr., 1913, LXXIX, 17) believes that eosino- 
philes gradually increase in the blood in favorable cases of tuberculosis; also, Brosamlen, 
Deutsch. Arch. f. klin. Med., 1914, CXV, 146; Peppard, Journal-Lancet, 1915, XXXV, 478. 

^Lifschiitz, Monatsschr. f. Kinderhkde., 1914, XII, 603; Aschenheim, Ztschr., f. Kin- 
derhkde., 1914, X, 503; Putzig, Ibid., 505; Engman and Davis, Jour. Cutan. Dis., 1915, 
XXXIII, 73; Brosamlen and Zerb, Deutsch. Arch. f. klin. Med., 1915, CXVIII, 163. 

^Schlecht and Schwenker (Deutsch. Arch. f. klin. Med., 191 2, CVIII, 405) show the 
relationship of eosinophilia to anaphylaxis. See Herrick, Arch. Int. Med., 1913, XI, 165; 
also, Ahl and Schittenhelm, Ztschr. f. d. ges. exper. Med., 1913, I, in; Steiger and Strobel, 
Zentralbl. f. inn. Med., 1913, XXXIV, 1073. Leopold (New York Med. Jour., 1914, C, 
225) finds an eosinophilia in chorea. Besancon and Moreau, Ann. de Med., I9i4> H, 85; 
Johns, New Orleans, Med. and Surg. Jour., 1914, LXVII, 453; Weinberg and Seguin, Ann. 
de rinst. Pasteur, 1914, XXIII, 470; Herrick, Arch. Int. Med., 1914, XIII, 794; Marcovici, 
Folia Haem., 1915, XX, 133. 

^ See Ebbell (Norsk Mag. f. Laegevidensk., 191 2, LXXIII, 617) for a very naive theory 
of the relation of eosinophilia to the parasitic diseases. Legueu and Morel, Jour, d' Urol., 
191 6, VI, 6c5, show the association of eosinophilia to enlarged prostate of non-malignant 
type. 



THE BLOOD 577 

are very frequently marked in the discharge in the early days of infection, 
diminishing in number as the number of neutrophiles increases, and increasing 
again as the discharge clears up. It is generally believed that the eosino- 
phile cells in the blood are increased coincidently with their increase in the 
gonorrheal discharge. It has been shown that the eosinophils are usually 
increased in all forms of ovarian disease with the exception of cancer. In 
syphilis a uniform increase of the eosinophiles has been observed, but some 
cases do not show such a regular finding. 

Outside of the splenomyelogenous form of leukemia and the true bron- 
chial asthma, infection with various parasites is accompanied by the most 
pronounced eosinophilia. Any parasite from the harmless pin-worm to 
the most malignant uncinaria may cause an eosinophilia (Emerson). This 
eosinophilia is not necessarily constant nor does its extent bear any relation 
to the severity of the infection. A differential diagnosis between typhoid 
fever and trichinosis is frequently possible on the basis of the marked eosino- 
philia in this latter condition,^ although eosinophilia does not always obtain 
here. 

Mast-cell Leucocytosis. 

In myelogenous leukemia we find these cells increased to a very large 
extent, often outnumbering the eosinophile cells. An increase of these cells 
is generally regarded as the sole isolated pathognomonic sign of this disease. 
Their increase may be as high as 20 per cent, of the leucocytes. These cells 
have been found in a few cases of cancer, tuberculosis, syphilis, and other- 
lesions of the skin, while in bone disease complicated by septic infection a few 
reports of positive findings have been made. 

Leucopenia. 

This is a condition characterized by a reduction either in the total number 
of leucocytes or in one or more groups of leucocytes. The most usual con- 
dition showing a leucopenia is typhoid fever in which we find the polynuclear 
cells markedly diminished, thus lowering the leucocyte count, while the large 
mononuclear cells are relatively increased. Anything below 5,000 cells is 
regarded as a leucopenia. It must be considered in this connection that the 
typhoid leucopenia remains only when the disease is limited to the intestinal 
canal; .when other organs become involved a leucocytosis supervenes. The 
count in typhoid fever may run as low as 2,000 cells, while in tuberculosis of 
the lymph-glands it may reach 500 cells. In cases of starvation or malnu- 
trition the leucocyte count is always low and in chronic intoxication with the 
heavy metals, morphin, alcohol, and cocain, we observe a very low count, as 
a rule. A general rule to be observed in typhoid fever is that an increase in 
the number of white cells following a low leucocyte count is evidence of com- 
plications or of a mistaken diagnosis. It must be remembered that a relapse 
in typhoid may bring on a hyperleucocytosis, but this must occur during the 

] , 
^ Cooper (Wisconsin Med. Jour., 1914, XII, 365) reports a case of trichinosis with 72 
per cent, of eosinophiles. 

37 



578 DIAGNOSTIC METHODS 

afebrile period, otherwise a leucopenia will remain.^ In measles, also, we 
observe a leucopenia following the eruption and a leucocytosis preceding the 
eruption. This leucopenia affects the polynuclear neutrophiles while the 
lymphocytes are relatively increased, with disappearance of the eosinophiles. 
In cases of uncomplicated influenza we usually find a diminution in the num- 
ber of leucocytes, although a normal number may obtain. This enables one 
to differentiate influenza from pneumonia, in which latter condition a marked 
leucocytosis is practically always present.^ In pernicious anemia and in 
splenic anemia we find a marked leucopenia during the active periods of the 
disease, the count running, as a rule, between two and three thousand cells. 

(F) Variations in Infancy and Childhood. 

In the first few days after birth the leucocytes may reach as high as 20,000, 
while in the nursing period the cells average 13,000. In this connection the 
influence of a digestion leucocytosis must be remembered as the cells may 
reach as high as 25,000 of which the lymphocytes constitute about 60 per 
cent. As the age of the child increases, the number of leucocytes gradually 
diminishes until the age of puberty, when the average number is about 8,000. 
As the development of the child progresses, the number of lymphocytes is 
gradually diminished and that of the neutrophiles correspondingly increased. 
The relations between the lymphocytes and the neutrophiles must be con- 
stantly remembered in making a blood count, as the percentage of lympho- 
cytes is high at first and then gradually diminishes until the age of pu- 
berty; the neutrophiles during the same period are at first low, reaching the 
normal 65 to 70 per cent, about the age of 15. There are no marked differ- 
ences in the morphology of the granular and of the nongranular cells of the 
child as compared with those of the adult. The characteristic changes in the 
leucocytes of the blood of the growing child are more related to the changes 
consequent upon development and show no signs of degenerative changes. 

(G) Functions of the Leucocytes. 

The functions of the leucocytes are largely related to their powers of 
overcoming the effects of bacterial processes. These results are accomplished 
both through their ameboid powers and their characteristic property of 
phagocytosis. This latter property is largely influenced by chemotaxis as 
well as by the indefinite increase of so-called opsonins. Just what we are to 
regard as the basis of the opsonic index of the leucocyte is unsettled, but it is 
certain that the same leucocyte may show marked variations in its power of 
absorbing bacteria of different types. Whether this opsonic power is related 
in any way to the various assumptions embraced by Ehrlich's side-chain 
theory must be left for a later section. Besides the above important func- 
tions of the leucocytes, these cells show oxidizing, reducing, and fermentative 
powers, all of which are of more or less importance in the study of immunity 

^ See Schneider (Deutsch. Med. Wchnschr., 19 15, XLI, 393 )for a discussion of leucopenia 
following typhoid inoculation. Ziersch, Miinch. med. Wchnschr., 1915, LXII, 1310; 
Austin and Leopold, Jour. A. M. A., 1916, LXVI, 1084; Wells, Jour. Infect. Dis., 1917, 
XX, 219. 

2 Camp and Baumgartner (Jour. Exper. Med., 1915, XXII, 174) show that severe 
inflammatory processes may be associated with a leucopenia. 



THE BLOOD 579 

and of reaction to bacterial infection. As stated (p. 557), the oxygen cata- 
lysts of the blood include oxidase, peroxidase, catalase and hemoglobin. 
To these ferments we owe many chemical tests for blood and pus, which are 
destroyed if the specimen be previously boiled, while hemoglobin is still 
capable of inducing certain oxidations. Kastle and Amoss^ have studied 
the peroxidase activity of the blood in health and disease, while Kastle^ has 
made an exhaustive study of the oxidases. Winternitz and Meloy,^ Winter- 
nitz and Pratt,* and, especially, Winternitz, Henry and McPhedran^ have 
turned their attention to catalase. The technic of these latter workers is as 
follows: 0.025 c.c. of blood is taken from a puncture of the ear by means of a 
specially graduated pipet. This is immediately diluted with 10 c.c. of dis- 
tilled water, giving a dilution of 1:400. Five c.c. of this diluted blood are 
placed in each of two 100 c.c. salt-mouth bottles, one being used for the test, 
the other being held as a check. In one is placed a small vial containing 
5 c.c. of neutralized commercial hydrogen peroxid (3 per cent.). The large 
bottle is connected with a gas buret, to collect the gas formed by the action 
of the catalase upon the H2O2. The bottle is agitated for one minute, read- 
ings being taken every 15 seconds. Eighty per cent, of the normal cases 
studied show a liberation of 14 to 17 c.c. .of oxygen in 15 seconds, this being 
constant for the same individual for a period of many months. The impor- 
tance of this test rests in the variations from day to day or in any change from 
the normal activity of the individual's blood. The amount of gas liberated 
is, therefore, a personal rather than a general factor. Tschernoruzki^ has 
recently shown that i gram of leucocytes is capable of liberating 312.9 c.c. 
of molecular oxygen under the influence of catalast. 

The diagnostic value of this test in disease is as follows: In typhoid fever 
there is no change in the early stages, but toward the third week there is a 
gradual fall accompanying the anemia. During the course of lobar pneumo- 
nia a slight decline may be noted. Diabetes mellitus shows no change. In 
diseases of the thyroid, the catalytic activity is not constant from day to day, 
in hyperthyreosis a tendency to increase being noted while in hypothyreosis a 
lower level than normal is observed. In renal disease we find no change as 
long as no marked renal insufficiency obtains, but the activity becomes irregu- 
lar from day to day, and, as uremia approaches, becomes much lower than 
normal. With retention of urine due to obstruction of the lower urinary tract 
a marked decline is observed. Little change is noted in cardiac cases even in 
the severe types. The toxemias of pregnancy are separable into two classes 

1 Bull. 31, Hyg. Lab. U. S. Pub. Health and Mar. Hosp. Serv., 1906. 

2 Bull. 59, Hyg. Lab. U. S. Pub. Health and Mar. Hosp. Serv., 1909. See Schultze, 
Miinch. med. Wchnschr., 1909, LVI, 167; Dunn, Jour. Path, and BacterioL, 1910, XV, 20; 
and Klopfer, Ztschr. f. exper. Path. u. Therap., 191 2, XI, 467; Evans, Arch. Int. Med., 

1915, XVI, 1067; Reed, Jour. Biol. Chem., 1916, XXVII, 299; Graham, Jour. Med. Res., 

1916, XXXV, 231. 

^Jour. Exper. Med., 1908, X, 759. 

* Jour. Exper. Med., 1910, XII, i and 115. 

5 Arch. Int. Med., 1911, VII, 624. See, also, Rohdenburg, New York Med. Jour., 1913, 
XCVII, 824; Waentig and Gierisch, Fermentforsch., 1914, 1, 165; Burge, Jour. Biol. Chem., 
1919, XXXVII, 343; Bodansky, Ibid., XL, 127; Reimann and Becker, Am. Jour. Physiol., 
1919, L, 54. 

^Ztschr. f. physiol. Chem., 1911, LXXV, 216. 



580 DIAGNOSTIC METHODS 

by this test; without renal involvement eclampsia shows no change but, when 
renal insufficiency obtains, a marked decline prevails. This test should be of 
much value in diagnosis. 

(7) Blood-plates. 

(a) Appearance. 

These bodies which have been called " third corpuscles" are probably not 
true cellular entities. Hayem has considered them as the direct forerunners 
of the erythrocytes and has styled them, therefore, "hematoblasts." These 
bodies have been variously called plaques by Osier, blood-plates by Bizzo- 
zero, and by Arnold fragments of cells. ^ These so-called corpuscles are small 
colorless bodies containing no hemoglobin. They are about 3 microns in 
diameter, round, oval, or rod-shaped without any biconcavity. They appear 
bluish, homogeneous or occasionally granular, and stained lightly by both 
basic and acid dyes. They do not contain any nucleus or membrane and 
become hyaline and glassy as soon as removed from the vessel, but on 
standing they become pale and unite to form granular masses. They are 
very sticky and adhere very extensively to one another, forming masses from 
which fibrinous threads radiate. This fact has lead to the belief that they 
have an important part in the formation of fibrin. (See Duke. 2) 

Specimens of the platelets are best obtained by puncturing the tip of the 
finger or the ear through a 10 per cent, solution of sodium metaphosphate. 
^ In this way the blood becomes at once mixed with the fixing fluid and the 
drop may then be placed on a slide and covered with a cover-glass. It has 
been customary to call anything a platelet which is smaller than a red-blood 
cell and which does not contain hemoglobin. The term platelet, however, 
should be reserved more particularly for these bodies which have a peculiar 
bluish refractility, no nucleus, show marked cohesive properties, and soon 
disintegrate. 

(b) Size. 

The normal size of the blood-platelet averages about 3 microns, although 
Preisich and Hein have reported them as high as 7 microns. As a rule, their 
size varies inversely as their number. Some of these bodies show clear areas 
either in the center or on one side, or on the whole periphery; others become 
crescents, triangles, spindles and threads (Emerson). 

(c) Number. 

The normal number of platelets per cmm. is approximately 250,000, this 
number varying in the same person at different times of day. The physio- 
logical factors influencing the number of these cells are not well understood 
and in pathological conditions we may find large or small numbers of these 
cells. It is generally accepted that they are increased in anemias from any 
cause and may be related to the red blood-cells as i to 10. They are usually 

^ See Downey, Folia Haemat., 1913, XV, 25; Brown, Jour. Exper. Med., 1913, XVIII, 
278; Winogradow, Folia, Haemat., 1914, XVIII, 207; Lee and Minot, Cleveland Med. Jour., 
1917, XVI, 65; Wright and Minot, Jour. Exper. Med., 1917, XXVI, 395; Emmel, Jour. 
Med. Res.,' 1918, XXXVII, 67. 

2 Jour. Am. Med. Assn., 1910, LV, 1185; also. Arch. Int. Med., 1912, X, 445. 



THE BLOOD 581 

normal in chlorosis, increased in severe secondary anemia and decreased in 
pernicious anemia. In splenomyelogenous leukemia we find a large increase 
of these bodies, while in the lymphatic leukemia these cells are diminished. 
They are increased in chronic diseases associated with cachexia and malnutri- 
tion, being more marked in cancer, nephritis and tuberculosis^ than in anemia, 
due to other causes. The more acute, more severe, more threatening the dis- 
ease, the fewer the number of platelets^ so that we have a direct relationship 
between severity of disease and number of these bodies. The method of 
counting these cells' has been given in a previous section to which the reader is 
referred. Just exactly what significance is to be attached to their increase or 
decrease is uncertain, but it must be remembered that there is a certain 
relationship between their number and the severity of general conditions. 

{d) Staining Properties. 

These bodies stain very much like nuclear material with a basic stain, but 
also take the acid dyes under certain conditions. With the Wright stain these 
cells are seen as distinctly blue bodies grouped in numbers from i to 10 and 
seem to be composed of nucleus and protoplasm. The apparent nucleus 
consist of rows of blue or reddish dots occasionally arranged in spherical 
masses, while the indefinite poorly-defined protoplasm-like substance seems 
swollen to almost the size of a red corpuscle. As a rule, however, these bodies 
appear as grouped bluish masses of indefinite structure and outline, 
(e) Function. 

The function of these cells is very indefinite. It is possible that they have 
much to do with the formation of fibrin and may be the source of the so-called 
fibrin ferment, thrombogen.^ If we are to regard these bodies as derived 
from the leucocytes this function of fibrin formation is acceptable, but if they 
are to be considered as derivatives of red cells or as true independent bodies 
such an hypothesis is untenable. No facts of any clinical value have so far 
been forthcoming from the study of the blood plates, and it may be possible 
that they are really artefacts as Lowit has claimed. 
(8) Hemoconien. 

By this term we have reference to the presence in normal blood of very 
fine granules which are actively motile, not truly ameboid, but with motion 
more of the Brownian type. These granules, also called blood-dust by Miiller, 
are small, round, colorless granules which vary in size from very fine dust- 
like particles to some as large as i micron in diameter. Their exact chemical 

^ See Webb, Gilbert and Havens, Arch. Int. Med., 1914, XIV, 743; Webb and Gilbert, 
Jour. Am. Med. Assn., 1914, LXIII, 1098. 

2 See Duke, Arch. Int. Med., 1913, XI, 100; also, Port and Akiyama, Deutsch. Arch. f. 
klin. Med., 1912, CVI, 362; Nobecourt and Maillet, Bull. Soc. de Pediat., 1914, XVI, 285; 
Weltmann, Wien. klin. Wchnschr., 1914, XXVII, 1013; Dimond, Jour. Path, and Bac- 
terioL, 1915, XIX, 508; Duke, Jour. A. M. A., 1915, LXV, 1600; Fonio, Cor. Bl. f. 
Schweiz. Aerzte, i9i5,XLV, 1505; Deutsch. Med. Wchnschr., 1916, XLII, 1344; Le Sourd 
and Pagniez, Ann. de Med., 1916, III, i; Minot and Lee, Arch. Int. Med., 19 16, XVIII, 
474; Minot, Ibid., 1917, XIX, 1062. 

^ Bayne- Jones (Am. Jour. Physiol., 191 2, XXX, 74) has shown that the platelets aid in 
the clotting of blood in two ways: (i) by setting free prothrombin, which is later activated 
to thrombin, and (2) by liberating a thromboplastic substance which neutralizes the anti- 
thrombin normally present in the blood. 



582 DIAGNOSTIC METHODS 

nature is uncertain as they do not show, according to Miiller, either the re- 
actions of fat or of albumin. The general idea prevails that these bodies are 
the extruded granules of the leucocytes, as they resemble in size and in stain- 
ing qualities those of various leucocytic types. The number of these gran- 
ules is uncertain, but apparently the relation of these granules to the red cells, 
as shown by the ultra condenser, is about 50 to i. 

Although no clinical significance is known to attach to these granules, the 
writer has observed marked variations in their number in various patholog- 
ical conditions, but cannot at present draw any conclusions from such 
observations. 

(9) Morphology of the Blood-forming Organs. 

As the bone-marrow is of such importance in the production of both red 
and white cells, it would seem advisable briefly to discuss the histology of 
this tissue. In it we find practically every cell which (Occurs in the blood both 
in normal and abnormal conditions, and also many transitional forms between 
various groups of cells. The following brief outline of the histology of the 
bone-marrow is taken from a valuable paper of Dickson.^ 

Varieties of Bone-marrow. 
(i) Primitive or Embryonic Marrow. 

A delicate interlacing network of mucoid cells, which later in the process 
of development go to form the connective- tissue framework or adenoid reticu- 
lum of the tissue. 

(2) Red "Lymphoid** or Formative Bone-marrow Proper. 

This is found in the adult in the short and flat bones, sternum, ribs and 
vertebrae, and to a varying extent in the ends of the long bones. This is the 
most important variety as in it are found the red and the majority of the 
white cells of the blood, and, according as one or the other of these series of 
cells predominates, the type of marrow may be classified as erythroblastic or 
leucoblastic, a varying admixture of these two types being practically always 
found in any given case. 

(3) Fatty or Yellow Marrow. 

This is found mostly, as age advances, in the central part of the long bones 
and is formed by a process of physiological transformation or degradation of 
the connective-tissue elements, together with the gradual disappearance of 
most of the blood-forming cells of the red marrow. 

(4) Fibroid Marrow. 

This is found in old persons, especially if debilitated by long-standing dis- 
ease, and is characterized by the proliferation of the connective-tissue ele- 
ments and by the progressive sclerosis of the marrow, followed by the gradual 
disappearance of the hemopoietic cells of the tissue. 

(5) Gelatinous Marrow. 

This is essentially a retrogressive change in the tissue and is in no way 
identical with that type already described as primitive or embryonic marrow. 

^ Jour. Path, and Bacteriol., 1907, XII, 136. 



THE BLOOD 583 

This change has been described by a previous writer as a chronic condition 
only, but has been frequently found by Dickson as an acute change in many 
of the acute infectious fevers and allied diseases. 

C3rtology of the Bone -marrow. 

In this discussion little more will be taken up than a brief enumeration of 
the various varieties of cells, as the more important ones of these have been 
treated of in other sections. 

(/) Blood-forming Cells. 

(A) Leucocyte Series. 

(a) Nongranular Cells with Basophile Protoplasm. 
(i) Large. 

(2) Small. 

(a) Cells similar to but smaller than the large variety. 
((3) Cells identical in appearance and in staining reactions with 
the small lymphocyte of the blood. Of these there are also 
probably two varieties. 

(b) Granular Cells. 
(i) Neutrophile. 

(a) Myelocytes with large rounded or oval nuclei. These have 
been definitely traced by Dickson to nongranular or hyaline 
cells in which the granules may be seen gradually develop- 
ing. There are two types known as the larger and the 
smaller neutrophile myelocyte. 

(|8) Intermediate cells with indented or horse-shoe-shaped 
nuclei. These are developed from the myelocytes (a) and 
in turn go to form 

(7) Polymorphonuclear cells or adult leucocytes which pass out 
into the blood stream. 

(2) Eosinophile. 

The same types of these cells with eosinophile granulations are found in 
the marrow, as have been previously tabulated under the heading of neu- 
trophile cells. 

(3) Basophile. 
(a) Mast cells. 

The three types above discussed are also observed in the 
basophile mast cells of the bone-marrow. 
(j8) Cells resembling the eosinophile myelocytes but with granu- 
lations staining with the basic dye. These cells are prob- 
ably altered eosinophiles. 

(B) Hemoglobin-holding Series. 

(i) Normoblasts Normocytes or ordinary red corpuscles, 

<' "i M 1 hi t / No^^nioblasts, normocytes. 

I Megalocy tes (entirely pathological in the adult) . 
(//) Giant Cells. 

(i) Mononucleated or megakaryocytes. 
(2) Multinucleated or polykaryocytes. 



584 DIAGNOSTIC METHODS 

(///) Cells of Connective-tissue Type. 

(i) Fat cells. 

(2) Cells of the reticulum. 

(3) Various forms of phagocytic cells. 

(4) Ordinary connective-tissue cells. 

{IV) EndotheUal Cells. 

(i) Found in their normal position in the vessel wall. 

(2) Found proliferating and taking on phagocytic functions. 

Reactions of the Bone-marrow in Disease. 

Many of these reactions are connected with the production of the so- 
called inflammatory leucocytosis and take place with great rapidity. Other 
varieties of change are intimately concerned with the production of the hemo- 
globin-holding series of cells. These reactions may, according to the type of 
cell involved, be summarized as follows: 
(/) Leucoblastic. 

(i) Neutrophile. (3) Basophile. 

(2) Eosinophile. (4) Hyaline or non-granular. 

(//) Erythroblastic. 

(i) Normoblastic. (2) Megaloblastic. 

The above brief outline will show the reader that we have in the marrow 
all possible types both of red and of white cells and that any variation in the 
normal activity of the marrow will result in the overloading of the blood with 
cells of a particular type depending upon the kind and extent of the affection. 

The histology of the other blood-forming organs, as the liver, spleen, 
and hemolymph nodes may be found in any text-book on histology. 

IV. Pathology of the Blood. 

(I) Special Pathology. 

Under the head of the special pathology of the blood we have to consider 
the conditions which are manifested directly by changes in the composition 
and cellular structure of this tissue. In very few of the blood diseases proper 
is the blood picture so characteristic that a definite diagnosis is always possi- 
ble, but in a few of them certain changes are more frequently found and more 
often lead to a presumptive diagnosis. The pathological conditions in the 
blood may be considered either primary or secondary, but it should be re- 
membered that severe secondary changes may so closely simulate those found 
in primary conditions that differentiation is almost impossible. It is probably 
true that all pathological changes of the blood are really secondary, but in a 
certain number of these states the etiologic factors are so obscure that we can 
do no more than interpret the blood findings as primary conditions. 

(A) Anemia. 

This is a condition characterized by a deterioration, both qualitatively and 
quantitatively, in one or in all of the blood constituents. It is usually char- 
acterized by a diminution in the percentage of hemoglobin (oligochromemia) 



THE BLOOD 585 

and by a decrease in the number of red cells (oligocythemia), but we should 
regard as essential factors in the anemic condition a reduction in the total 
volume of blood (oligemia) as well as a reduction in the amount of protein 
(hypalbuminosis) . As much more attention has been paid to the first two 
factors than to the latter ones, anemia has come to mean a reduction in the 
amount of hemoglobin with a more or less extensive reduction in the number of 
red cells. Associated with these conditions we have, in the severer types of 
anemia, variations in form, size, and structure of the red cells as well as defi- 
nite changes in the relationship of the different white cells. In the study of 
the anemic conditions we must differentiate the primary from the secondary 
form, by this we mean a .differentiation between those forms which have no 
demonstrable cause from those types whose etiology is more or less secondary 
to other pathologic conditions. 

Primary Anemias. 

(i) Simple Primary Anemia. 

This form which has no demonstrable cause is difficultly separable from 
the secondary form as well as from certain other primary forms, such as pri- 
mary pernicious anemia. It will probably be shown to be a true secondary 
anemia and must be sharply differentiated from the pernicious type from the 
point of prognosis. These cases are only recognizable when they are typical 
in form and are frequently not amenable to any form of treatment which we 
may institute. The rule, however, is that these forms of anemia yield more 
or less promptly to proper dietetic and therapeutic treatment. It is probable 
that the question of prognosis in these cases must depend largely upon the 
amount of degeneration of the red cells which occurs.^ In these cases we find 
that the diminution in the number of red cells is usually parallel to the re- 
duction in the amount of hemoglobin, so that a high color index will usually 
obtain. More or less degeneration, as evidenced by the appearance of poi- 
kilocytes, normoblasts, Maragliano's polychromatophilia, etc., will be 
observed depending upon the severity of the case. The leucocytes are usually 
normal in number and in differential relations,_while the blood plates are 
usully increased. The changes in the plasma in this primary anemia are 
not characteristic, although we do observe a diminution in the specific 
gravity which runs parallel to the oligochromemia. 

(2) Chlorosis. 

Chlorosis is a primary anemia occurring almost exclusively in girls about 

the age of puberty and characterized by a marked reduction in the amount 

of hemoglobin and a slight change in the number of red cells. Clinically, this 

state is evidenced by the appearance of a wax-like changing into a greenish 

tone of the skin and a sky-blue coloration of the cornea. Some cases may 

show a variety of colors of the skin. Certain changes are observed in the 

digestive and generative organs and certain general abnormalities are seen 

^ See Pollitzer (Ztschr. f. klin. Med., 191 2, LXXV, 367) for a discussion of the types of 
regeneration and degeneration in anemia; also, Wichern and Piotrowski, Deutsch. Arch. f. 
klin. Med., 191 2, CVI, 533; Pribram, Deutsch. Arch. f. klin. Med., 1914, CXVI, 535; 
Kleinschmidt, Jahrb. f. kinderhkde., 1916, LXXXIII, 97. 



586 DIAGNOSTIC METHODS 

which are due to lessened production of blood-cells and to diminished oxida- 
tive and fermentative powers of the system. This form of primary anemia 
differs from all other forms in the absence of blood degeneration, as very 
rarely marked degenerative signs appear in the blood picture. The blood 
finding is not absolutely characteristic for this clinical entity, as it is simu- 
lated by many anemias of the secondary type. Clinically, this disease is 
so sharp that a diagnosis is often possible without a blood examination. 

The chief characteristics of the blood in this condition are: (i) Reduction 
in the hemoglobin. This may run as low as 20 per cent., giving a color index 
of 0.5. Secondary anemias rarely reach such a low level. (2) Variations in 
the number and size of the red cells. The number of red cells is not reduced 
to a very great extent, the average being about 4,000,000, although counts as 
low as 1,000,000 have been reported. When these low counts do occur some 
complication should be suspected. Ordinarily the size of the cell is diminished, 
although we frequently find large "dropsical" cells which are due to 
absorption of fluid from the hypotonic plasma. These latter cells are usually 
few in number, the great majority of cells being smaller than the normal size. 
Poikilocytes and degenerated reds rarely occur except in the severer forms of 
this disease, while chromatophilia is usually regarded as a sign of active re- 
generation of the blood. When nucleated reds occur, which is a rare find- 
ing, they are practically always of the normoblastic type and rarely appear of 
the megaloblastic form. 

The leucocytes in this condition are usually normal both in size and 
number and degenerative forms are rarely seen. It has been stated that the 
eosinophile cells are much increased in this condition, but the writer has 
found that their ratio is very rarely above the upper limit of the normal 
figure for these cells. The platelets are usually about normal and are usually 
large in size. 

In chlorosis we find certain variations in the physical and chemical prop- 
erties of the blood. The specific gravity is usually reduced in proportion to 
the reduction of the hemoglobin and may fall as low as 1028. The plasma is 
diluted so that a condition of oligemia may be considered more or less 
characteristic of chlorosis. Whether or not a hydremia, as indirectly mani- 
fested by a diminution in the amount of albumin, obtains, is debatable.^ 

Chlorosis belongs to the class of primary anemias and as such has no 
definite etiology. Various conditions such as hypoplasia of the arterial 
system, intestinal autointoxication, disturbances of the nervous system, such 
as a vasomotor neurosis, have been advanced to explain this condition, but 
none of them are tenable in all cases. A great importance must attach to 
hygienic conditions, poor food, and mental depression, especially about the 
age of puberty, as this disease is usually apparent under these conditions at 
this time of life. A further factor which must necessarily bear upon the 
etiology of this condition is the defective power of absorption of iron com- 
pounds. Patients afflicted with chlorosis improve rapidly under the ad- 
ministration of iron, but not unless the digestive and absorptive powers im- 

^ See Frohmaier, Folia Haem., 191 5, XX, 115. 



PLATE XXTII. 










D «^ 



0^^ 










'^v? 



^^8 



0' 



^ c^^ 



c^ 



1,0 




5_ r.-i- •,, oi- ,^5 



'i^ ^*'<:^ 



Chlorotic Anemia. (Wright's Stain.) 



THE BLOOD 587 

prove at the same time. It does not concern us here as to the dynamics of 
the absorption and the effect of iron, but it should be accepted as an axiom 
that no therapeutic effects may be expected from iron unless the absorptive 
power is made better. Just exactly what the pathological conditions are 
which are accountable for the functional insufficiency of the bone-marrow is 
uncertain, but it must be recalled that gross as well as microscopic pathologic 
changes are not necessary to produce functional disturbance in any organ. 

It should be remembered that we may have various types of chlorosis 
which show different prognostic characteristics: (i) Those in which the red 
cells are very slightly reduced (about 4,000,000), a marked diminution in the 
amount of hemoglobin, a low color index, and no change in the size and shape 
of the cell. Such cases usually recover promptly without showing any re- 
lapse. (2) Cases in which the red cells are below 4,000,000, which show a 
marked diminution in the amount of hemoglobin with a very low color index 
and which give very slight evidences of degenerative changes in the red cells. 
These cases are usually characterized by marked prostration, but usually re- 
cover more or less promptly although relapses frequently occur. (3) Cases 
in which the red cells are reduced as low as 2,500,000, a reduction in the 
hemoglobin with a very low color index, marked changes in the shape and 
size of the red cells. These cases respond slowly to treatment and have a 
bad prognosis (Ewing) . 

As these cases of chlorosis convalesce, we observe an increase in the 
number of red cells to a point somewhat above the normal and a later increase 
in the hemoglobin content of each individual cell. These changes are usually 
evident in from eight to ten days after institution of treatment, but as a rule 
a much longer time is necessary for any marked change to be observed in the 
number of the cells or in their hemoglobin content. The changes in the 
plasma are usually the first to appear and should be considered essential for 
the proper regeneration of the blood in chlorotic conditions. 

This disease belongs in the group of curable conditions and usually has a 
good prognosis, but the susceptibility to intercurrent acute diseases during 
this period is much increased so that we should always bear in mind the 
probability of complications arising in the convalescent period. 

(3) Progressive Pernicious Anemia (Biermer's Anemia). 

This term is applied to a form of severe anemia which, in spite of all 
treatment, progresses steadily toward death. It results from defective hema- 
togenesis and excessive hematolysis and is characterized by definite changes, 
both numerically and morphologically, in the red cells and by characteristic 
changes in the bone-marrow (Ewing). This condition has frequently been 
described as the result of infection with certain intestinal parasites so that it 
cannot in all cases be considered a truly primary disease, although the larger 
majority of cases show no known etiology. ^ According to Herter, certain 
anerobic bacteria, found in the large intestine, produce a substance of marked 

^ See Friedstein, Inaug. Dissert., Berlin, i9i2;Ludke and Fejes, Deutsch. Arch. f. klin. 
Med., 1913, CIX, 433; also Pilcher, Am. Jour. Med. Sc, 1913, CXLVI, 226; Ragosa, Folia 
Haemat., 1915, XIX, 269. 



500 DIAGNOSTIC METHODS 

hemolytic power, which penetrates the intestinal wall and enters the portal 
circulation.^ In some cases of idiopathic purpura hemorrhagica the blood 
picture is that of pernicious anemia, but may be distinguished by the absence 
of megaloblastic change and by the prominence of hemorrhage. ^ The blood 
picture in this disease is not absolutely characteristic, as certain forms of 
secondary anemia may show similar findings. The chief characteristics of 
the blood in primary pernicious anemia, as also in those severe types of 
secondary anemia which simulate this form, are (i) signs of rapid blood de- 
struction, such as degenerated reds, endoglobular degenerations, polychroma- 
tophilia, increased iron compounds in the serum and corpuscles, an increase 
of iron in the liver and spleen ; (2) poikilocytosis; (3) a high color index result- 
ing from a marked diminution in the number of red cells and a correspondingly 
less degree of diminution in the amount of hemoglobin; (4) megaloblastic 
blood formation. This latter indicates a direct reversion to the embryonal 
type of blood formation in which the presence of megaloblasts as direct pre- 
cursors of megalocytes is observed.^ The red blood-cells in this condition 
are few in number being reduced to as low as 1,000,000 cells, counts of 
500,000 having been observed without the patient suffering any marked in- 
convenience. Naegeli reports a case showing 138,000 reds. This fact 
should be taken as evidence that the oligocythemia in pernicious anemia is 
not alone accountable for the symptomatology. This count may remain 
stationary, may show slight decrease, but usually progresses slowly until 
death ensues. The average diameter of the red cells is somewhat increased in 
pernicious anemia. While many of them may be normal in size and many 
very small, the cells average from 4 to 13 microns in diameter. A pernicious 
anemia is a distinct large cell anemia, A macrocy tosis is much more character- 
istic of this disease than of any other, 70 per cent, of the cells in these cases 
being of this type.^ Microcytosis is rare but may occur to such an extent 
that the average size of the red cell may be about normal. Poikilocytes are 
very common and often show extreme shapes and are frequently numerous in 
number. Polychromatophilic degeneration is very extensive in this form of 
anemia. 

While both normoblasts and megaloblasts occur in large numbers in per- 
nicious anemia, the megaloblasts usually outnumber the normoblasts. This 
megaloblastic increase may be considered pathognomonic of this anemia, as a 
preponderance of these large nucleated red cells does not occur, except in rare 
cases, in the other varieties of anemia. The hemoglobin may be markedly 
reduced showing values rarely above 50 per cent, and often as low as 10 per 
cent. The color index is always high; being more frequently above one than 
below. A low index is found, according to Ewing, in the chronic cases, while 
the acute forms are more frequently associated with a high index. If im- 

1 See Banti, Semaine med., 1913, XXXIII, 313; also, Robertson, Jour. Biol. Cham., 
1915, XVI, 652. 

2 See Minot, Am. Jour. Med. Sc, 1916, CLII, 48. 

3 See Brosamlen, Deutsch. Arch. f. klin. Med., 1913, CXII, 83; Vogel and McCurdy, 
Arch. Int. Med., 1913, XII, 707; Roth, Ztschr. f. klin. Med., 1914, LXXIX, 266. 

^See Diinner, Berl. klin, Wchnschr., 1914, LI, 1759. 



PLATE XXIV. 




Blood in Pernicious Anemia. (Wright's Stain.) 



THE BLOOD 589 

provement occurs the index is always lowered, an increasing index denoting 
a bad prognosis. 

In this pernicious form of anemia we find the leucocytes practically al- 
ways diminished in number, averaging about 4,000, a condition which almost 
never obtains in a secondary anemia. Their number usually runs parallel to 
that of the red cells, a leucocytosis pointing probably to a complication. As 
the case improves the neutrophile cells increase in number, the low leucocyte 
count being, as a rule, due to their diminution. The percentage of the non- 
granular mononuclear cells varies inversely to that of the granular form. As 
the disease progresses the percentage of the nongranular cells increases, while 
the granular cells are diminished. This disease shows, therefore, a high lym- 
phocyte count which is relative and not real, being due to the diminution in 
number of the polymorphonuclear cells. The lymphocytes may constitute 
as high as 50 per cent, of the leucocytes, the eosinophiles may reach as high 
as 10 per cent., the myelocytes 2 per cent., while the mast cells may run as 
high as 3 per cent. Degenerations of all kinds are observed in the leucocytes, 
but nothing characteristic of pernicious anemia is found in the white 
cells. ^ 

The blood-platelets are largely decreased and may be as low as one- 
twenty-fifth of the normal number. Von Limbeck and Sahli claim that these 
cells are increased in number, but the usual finding is one of diminution. 

(4) Splenic Anemia. 

This is a form of chronic anemia characterized by idiopathic enlargement 
of the spleen without any involvement of the lymph nodes. A large number 
of conditions may be responsible for the anemia of the splenic type, so that a 
direct etiologic factor should be looked for in all cases. Among the conditions 
which may give rise to this type of anemia, we find gummata of the spleen, 
large round-cell sarcoma of the spleen, chronic splenitis of the malarial type, 
and splenomegaly associated with cirrhosis of the liver. This latter con- 
dition is known as Banti's disease and its etiology is uncertain.- 

While it is true that the splenic lesions do not differ essentially from 
certain stages of the lesions in ordinary pseudoleukemia, yet we do not find 
in this condition any involvement of the lymph-glands, although the spleen 
may be enormously enlarged. The blood picture in this condition is character- 

1 See Nicol, Deutsch. Arch. f. klin. Med., 1913, CXI, 417; Sisto, Policlinico, 1913, XX' 
509; Ibid., IQ14, XXI, 34, 91 and 115; Wolff, Deutsch. Med. Wchnschr., 1914, XL, 643' 
Cederberg, Berl. kHn. Wchnschr., 1914, LI, 585; Mikhailoff, Russk. Vrach., 1914, XIII' 
837; Cavaglieri, Gazz. d. osp., 1914, XXXV, 1169; Schmidt, Am. Jour. Med. Sc, 1914'. 
CXLVIII, 313; Briggs, Ibid., 413; Moffitt, Ibid., 817; Krumbhaar, Ibid., 1915, CL, 227' 
Drinker and Hurwitz, Arch. Int. Med., 1915, XV, 733; Vogel, Jour. A. M. A., 1916, LXVI' 
1012; Schneider, Arch. Int. Med., 1916, XVII, z^] Roccavilla, PoHclinico, 1916, XXIII, 
281, 329. 367 and 399; Esch. Ztschr. f. Geburtsh. u. Gyn., 1916, LXXIX, i; Jessen and 
Unverricht, Miinch. Med. Wchnschr., 1916, LXIII, 1787; Giffin, Jour. A. M. A., 191 7, 
LXVIIT, 429; Squier. Jour. Lab. and Clin. Med., 1917, II, 552, Barsky and Kahn, Proc. 
Soc, Exp. Biol, and Med., 1918, XVI, 31; Kahn and Barsky, Arch. Int. Med., 1919, 
XXIII, 334; Rombach, Nederl. Tijdschr. v. Geneesk., 1919, II, 671. 

2 See Banti, Berlin, klin. Wchnschr., 1911, XL VIII, 2328; Stein, Am. Jour. Med. Sc, 
1912, CXLIV, 856; also. Umber, Miinch. med. Wchnschr., J91 2, LIX, 1478; Lacouture, 
Duperie, and Charbonnel, Jour, de med. de Bordeaux, 1913, LXXXIV, 805; Caronia, 
Pediatria, 1914, XXII, 752; Sailer, Penn. Med. Jour., 1914, XVIII, 91; Krull, Mitt. a. d. 
Grenzgeb. d. Med. u. Chir., 1914, XXVIII, 718. 



590 DIAGNOSTIC METHODS 

ized by a relatively high red cell count, a marked reduction in the percentage 
of hemoglobin, and a consequent low color index. The white cells are rarely 
increased, a leucopenia being rather the rule. When we do find an increase 
in the number of white cells a relative lymphocytosis occurs usually associated 
with an increase in the number of basophiles. Poikilocytes and nucleated red 
cells are very uncommon while degeneration may occasionally be observed. 
Whether or not we have an increase or a decrease in the number of leucocytes, 
a relative lymphocytosis associated with enlarged spleen and no involve- 
ment of the lymph nodes must be considered characteristic of splenic 
anemia. 

In Banti's disease we find an enormous increase in the size of the spleen 
and associated with this an extensive cirrhosis of the liver. Along with 
these factors we find a high-grade toxogenic protein decomposition, which is 
associated with very high values for the total nitrogen of the urine and of the 
output of purin- bases. The number of erythrocytes diminishes correspond- 
ing to the degree of anemia, while the hemoglobin percentage is more markedly 
reduced. The leucocytes are either normal or more frequently diminished 
in number, a relative lymphocytosis existing as in the pure tyipe of splenic 
anemia. 1 

(5) Anemia Infantum Pseudoleukemica. 

Von Jaksch has described a rare form of anemia seen in children which 
is characterized by enlargement of the spleen, liver, and lymph nodes. The 
most striking points in this condition are the great diminution in the number 
of red cells, one case showing only 820,000; numerous nucleated red cells; 
diminution of hemoglobin; the leucocytes always increased in number, being 
from 20,000 to 50,000, as a rule, and displaying a remarkable variety of form 
and frequently attaining to unusual size. The morphological changes in the 
blood resemble both those seen in leukemia and in pernicious anemia, the dis- 
ease passing either into one or the other of the previously mentioned con- 
ditions. The blood findings are not alone sufficient to warrant a diagnosis of 
infantile pseudoleukemia, but are significant when taken in conjunction with 
the clinical findings.^ 
(6) Leukanemia. 

This condition has been classed by some with the leukemias as the blood 
findings are occasionally more prominent among the leucocytes, while by 
others it is classed with pernicious anemia owing to the frequent changes in 
the red cells. Von Leube considers this anemia a mixed form of pernicious 
anemia and of leukemia. Luce^ regards this as a symptom of many conditions 

^ Yatfes, Bunting and Kristjanson (Jour. Am. Med. Assn., 1914, LXIII, 2225) report the 
isolation of pure cultures of Bacillus hodgkini (see Chap. XI) in two cases of Banti's disease. 
They beUeve there is an etiologic relationship here. See, also, Gibbons (Quart. Jour. Med., 
1914, VII, 153), who reports the presence of a streptothrix in six cases. Paisseau and 
Lemaire, Arch, des Mai. du Coeur, 1916, IX, 473; Humphrey, Allbutt, Deighton and Hare, 
Brit. Med. Jour., 1916, I, 365; Eberly, Jour. A. M. A., 1916, LXVII, ss'i Joseph, Ibid., 
1936; Norris, Symmers and Shapiro, Am. Jour. Med. Sc, 191 7, CLIV, 893. 

2 See Stillman, Am. Jour. Med. Sc, 1917, CLIII, 218; Kilduffe, Arch. Diag., 191 7, X, 6. 

•■'Deutsch. Archiv. f. klin. Med., I900, LXXVII, 215. See, also, Martelli, Virchow's 
Arch., 1914, CCXVI, 224; Gazz. internat. di med., 1914, XVII, 553 and 577. Allan and 
Leinbach, Jour. A. M. A., 1917, LXVIII, 1020. 



PLATE XXV. 




Blood in Leukanemia. (Wright's Stain.) 



THE BLOOD 59I 

rather than an independent blood disease. In the majority of these cases the 
changes are more evident in the red cells, marked diminution in number (as 
low as 200,000 per cmm.) associated with extensive destruction of red cells 
with all irregular and unusual types of these cells being observed. The dim- 
inution in the amount of hemoglobin is great, but the color index is usually 
high just as we find it in pernicious anemia. The white cells show an exten- 
sive disturbance in the neutrophile and eosinophile blood picture with a 
large increase in the number of large lymphocytes. The number of white 
cells is usually increased, but not to as great an extent as in leukemia. The 
changes in the red cells and in the hemoglobin usually precede those in the 
white cells, so that the early stages may show us a typical picture of pernicious 
anemia, while later examinations may lead to the diagnosis of the mixed 
condition. 

(7) Aplastic Anemia. 

Ehrlich has reported a rapidly progressing anemia accompanied by 
hemorrhages into the mucous membranes, associated with hyperplasia of 
the bone-marrow, and not showing the ordinary changes in the blood which 
are supposed to accompany pernicious anemia. This type has been called 
aplastic anemia and has not been frequently reported. 

The red cells are usually markedly reduced in number, being as low as 
790,000 in a case reported by Wood. The hemoglobin may fall as low as 
1 1 per cent, as reported by Muir, while the leucocytes are usually normal in 
number but showing much reduced percentages of the polynuclear neutro- 
philes. In this condition we find an enormous increase in the number of 
lymphocytes, the percentage being in Lipowski's case 93, the remaining 7 
per cent, being neutrophiles. No nucleated reds have been found in the 
blood and only a very few in the marrow itself, which is fatty, almost white 
and contains few neutrophiles and no eosinophiles.^ 
Secondary Anemia. 
By secondary anemia we mean one in which etiological factors seem 
sufficient to explain the variations observed in the blood. The principal 
variation seems to be more directly observed in the reduction of the hemo- 
globin, although the number of red cells is coincidently reduced, but not 
to the same degree as is the hemoglobin. In the mild cases the color of the 
blood is but slightly paler than normal, but in secondary anemia of a severe 
type the color may resemble the watery drop observed in cases of typical 
pernicious anemia. 

Cabot suggests a classification of the secondary anemias as follows: 
(i) Mild cases, showing a normal count of red cells, but having the hemo- 
globin diminished. (2) Moderate cases in which the count is normal but the 
cells show signs of moderate degeneration, abnormal staining qualities, and a 
diminished tendency to rouleaux formation. (3) Severe cases, in which the 
count is not much reduced but in which the hemoglobin is very much lessened 

^ See Hirschfeld, Folia Haemal., 1911, XII, 347; Musser, Arch. Int. Med., 1914, XIV, 275; 
Kleinschmidt, Jarb. f. Kinderhkde., 1915, LXXXI, i; Predtechensky, Russk. Vrach, 
1915, XV, 313; Frank, Berl. klin. Wchnschr., 1915, LII, 961 and 1062; O'Malley and Con- 
rad, Jour. A. M. A., 1919, LXXIII, 1761. 



592 DIAGNOSTIC METHODS 

and the cells show marked qualitative and quantitative changes. (4) Very- 
severe cases with a slightly lessened blood count, a marked diminution of the 
hemoglobin, and evidences of degeneration and destruction of the cells as well 
as evidence of regeneration. In some of the severer types of secondary ane- 
mia the blood picture may so closely resemble that of pernicious anemia that a 
diagnosis is possible only through the careful investigation of the etiology 
of the condition. 

Blood Picture. 

The general points observed in secondary anemia are as follows: A vari- 
able decrease in the amount of hemoglobin, a reduction in the number of red 
cells less in degree than the diminution of hemoglobin and a subnormal 
color index, which rarely reaches the low grade shown in chlorosis. The 
lowest color index is seen in those secondary anemias following cancer, severe 
hemorrhage, and gangrenous processes. The specific gravity of the blood is 
reduced corresponding to the degree of reduction of hemoglobin. The rapid- 
ity of coagulation is increased depending upon the grade of oligochromemia 
and of oligocythemia. The reduction in the number of red cells may be very 
marked as, for instance, in a case of von Limbeck the number was 306,000. 
The red cells show a lack of hemoglobin, frequently appearing as the pessary 
forms. Polychromatophilia is quite common, but bears no direct relation to 
the hemoglobin content of the cell. Only in the severer cases do we find 
poikilocytes, although anisocytes, especially microcytes, are frequently pres- 
ent, the larger "dropsical" cells being less common than in chlorosis. Nu- 
cleated erythrocytes are frequently seen in some cases, while in others even 
of severer grade of anemia they are absent. These nucleated reds are, as a 
rule, of the normoblastic variety, megaloblasts being exceedingly rare. 

The leucocytes vary in number depending on the cause of the anemia, 
from a leucopenia, which is rare, to a leukemic condition. The increase in the 
number of leucocytes is more frequently in the polynuclear neutrophiles, the 
lymphocytes being rarely if ever increased, while the eosinophiles, although 
not increased, are usually at the upper limit of the normal value for these cells. 
In some cases of the severe chronic types of secondary anemia we may find a 
lymphocytosis, but this is rare. The blood-platelets are usually increased in 
number, in some cases being two and one-half times the normal values, 
(i) Acute Hemorrhage. 

The character of the anemia following an acute hemorrhage will depend 
upon the type of the hemorrhage, that is whether the loss of blood occurred 
suddenly and at one period, or whether slowly and at intervals.^ The loss of 
one-half of the total volume of blood at one time is usually fatal, as Panum has 
shown. If the loss be less following one large hemorrhage, regeneration takes 
place in from five to thirty days, depending upon the amount of blood lost. 
Regeneration is quickest in men between the ages of 20 and 40, slower in 
women, and slowest in children. 

Immediately following a hemorrhage the blood picture will be normal 

^ See Milne, Jour. Exper. Med., 1912, XVI, 325; Deutsch. Arch. f. klin. Med., 1913, 
CIX, 401. 



THE BLOOD 593 

qualitatively as there has been no time for the morphologic changes to take 
place. Shortly following the hemorrhage lymph pours into the blood to re- 
store the volume and maintain the pressure, so that the blood count and the 
hemoglobin diminish usually to the same degree. As the formation of new 
cells goes on the color index decreases, as the new cells are, as a rule, deficient 
in hemoglobin. These cells are more or less easily degenerated, showing varia- 
tion both in their shape and staining qualities. The number of cells reaches 
the normal much sooner than does -the percentage of hemoglobin, so that we 
may find for weeks evidences of marked anemia. 

The most frequent causes of acute hemorrhagic anemia are traumatism, 
ectopic pregnancy, abortion, gastric and duodenal ulcers, uterine tumors, pul- 
monary tuberculosis, and hemorrhagic pancreatitis. 

(2) Chronic Hemorrhage. 

By a chronic hemorrhage we mean one in which repeated hemorrhages 
follow one another so closely that the blood has no time to regenerate before 
a second loss of blood occurs. This rules out of consideration those cases in 
which repeated hemorrhages occur but at sufficiently long intervals to permit 
of regeneration of the blood. This latter condition, although a chronic 
hemorrhage, gives the picture described under Acute Hemorrhage. 

In the chronic hemorrhage we find the red cells markedly reduced, the 
hemoglobin very much diminished and usually a marked leucocytosis. The 
red cells are usually small and pale, show a low color index, and usually few 
nucleated forms, although the picture may rarely assume the pernicious type. 
Regeneration in this form of anemia is slow, as the blood-forming organs seem 
to lose their power of regenerating the blood after repeated hemorrhages. 

The most frequent causes of chronic hemorrhage are scurvy, epistaxis, 
hemorrhoids, intestinal ulcers, gastric and other carcinomata, and intestinal 
parasites. 

(3) Inanition. 

In the discussion of the anemia of inanition, it must be remembered that 
other factors than starvation are necessary in its causation. That starvation 
alone will not cause anemia is shown from the examination of the blood of 
Cetti who fasted ten days. His blood showed about 6,000,000 red cells, a 
small diminution in the percentage of hemoglobin, and a leucocyte count of 
4,200. Although the changes in the cells and the pigment of the blood are not 
marked following starvation, it is true that we have a loss of albumin of the 
plasma and a diminution in the total volume of blood. This is taken as 
evidence by Grawitz that a true anemia occurs. Such changes are not strik- 
ing if the days of fasting are alternated with days of slight nourishment. It is 
not so much the quantity of food as the quality which is of importance in 
bringing about the anemic conditions. As is well known, the foods containing 
iron are the principal sources of the hemoglobin of the blood, and these are 
frequently, owing to disturbed gastric and intestinal functions, poorly digested 
and assimilated. Although the amount of iron contained in the ordinary 
food is sufficient under the best conditions to maintain the hemoglobin con- 
38 



594 DIAGNOSTIC METHODS 

tent of the blood, yet the methods of food preparation, as well as the abnor- 
mal methods of rapid eating, are important factors in the poor assimilation of 
the iron of the food. 

The lack of sunlight as well as impure air are contributory factors in 
causing anemia through their influence upon the general body functions. No 
tissue, whether animal or plant, can flourish in air which does not have 
sufficient oxygen to support the combustion processes of the system. This 
statement needs no retraction in the case of anaerobic bacteria, as it has been 
definitely shown that these organisms obtain the oxygen necessary for their 
development from the culture media upon which they grow, although they are 
incapable of developing in an atmosphere of pure oxygen. Overwork, espe- 
cially when associated with worry, has great influence upon all of the func- 
tions of the system. For this reason overwork has been credited with the 
power of producing anemia as well as many other serious systemic disturb- 
ances. However, it is rare to find an authentic case of anemia which can be 
traced directly to overwork without the mental influence of worry and the 
coincident nervous strain from this latter cause. ^ 

(4) Intestinal Parasites. 
The anemia caused by intestinal parasites may be of such a severe grade 
as to resemble very closely the type of pernicious anemia. In many cases it 
is an impossibility to make a differential diagnosis between these types with- 
out the finding of an intestinal parasite which will clear up the diagnosis. In 
these cases the blood picture returns more or less quickly to normal after re- 
moval of the parasite in question, while in the pernicious anemia of unknown 
origin the progress of the disease is always toward a fatal termination. It is 
probable that the cause of the severe secondary anemia due to the presence 
of the intestinal parasite is a result of the toxic condition set up by the ab- 
sorption of the hemolytic toxins elaborated by the parasite. A very severe 
anemia of the secondary type is frequently seen as a result of decomposition 
of the intestinal contents and in cases of chronic constipation in which the 
direct toxic agent is at present unknown. The most common intestinal para- 
sites causing the severe types of secondary anemia are (i) uncinaria duode- 
nale, (2) strongyloides intestinalis, and (3) bothriocephalus latus. The first 
of these causes an anemia which is very closely related to that shown by 
miners and tunnel diggers, and seems to be much more prevalent in the 
southern part of the United States, although it occurs in many different 
countries. The blood count may fall below one million red cells and the 
hemoglobin may be as low as 15 per cent., while all varieties of degenera- 
tive changes may be seen in the erythrocytes. In the anemia following 
infection with the bothriocephalus we find very marked similarity with the 
primary pernicious type. One-half to two-thirds of the nucleated reds in 
this variety may be of the megaloblastic type and yet may disappear within 
two to three weeks after the worm has been expelled. It is uncommon to 
find in secondary anemias caused by the parasites above mentioned any 
marked eosinophilia, which is so common in cases of infection with many of 
1 See Ash, Arch. Int. Med., 1914, XIV, 8. 



THE BLOOD 595 

the other forms of intestinal parasites. The anemia of the severer type 
seems to prevent a chemotaxis toward eosinophile cells. 

(5) Fever. 

It is still very much of a question whether the blood in febrile cases shows 
the characteristics of a secondary anemia, as the result of the temperature 
increase. It has been shown that increased temperature, in itself, does not 
always produce anemia, although we do have marked destruction of the red 
cells and .a coincident loss in the amount of hemoglobin of the remaining 
cells. So great is the influence of the toxin of the febrile condition that it is 
highly probable that the anemia so frequently observed in febrile cases is due 
to a combination of causes, rather than to a specific effect of the increased 
temperature. The changes in the white cells in febrile cases are not always of 
the same character, the variations being dependent upon the specific causative 
factor of the fever. As the blood changes in the acute infectious fevers are of 
more or less importance, they will be discussed later under separate headings. 
The anemias which are secondary to both the acute and chronic infections 
are probably directly due to the influences of the toxins upon the blood and 
blood-forming organs. The condition of general nutrition as well as the 
state of digestion, especially in the chronic states, such as tuberculosis, 
leprosy, and syphilis, must be regarded as important factors in the causa- 
tion of these secondary types. 

(6) Blood Poisons. 

There are a very large number of compounds which produce, when taken 
in toxic doses, very marked changes in the qualitative and quantitative com- 
position of the blood. As is well known, iron compounds in therapeutic doses 
increase the amount of hemoglobin in the red cells and also increase the num- 
ber of red cells up to a certain point. Many of the effects which are attrib- 
buted to iron compounds may be due to the improved hygienic and dietetic 
conditions which usually prevail during the administration of these sub- 
stances. Yet the therapeutic results following the administration of iron 
are such as to make it certain that a specific influence of this drug is present 
in anemic conditions, especially of the chlorotic type. 

Many compounds produce a very marked secondary anemia, the most 
important of these being alcohol, opium, lead compounds, cocain, and ace- 
tanilid. While others, such as arsenic, nitrobenzol, nitroglycerin, phenacetin, 
and poisonous mushrooms, cause dissolution of the red cells with marked 
hemoglobinemia. The anemia following the use of lead, either in toxic doses 
or after its slow absorption from constant contact with it in the arts, is of 
great practical importance. The causes of this lead anemia are rather com- 
plex. The lead compounds have a direct action on the red cells and on the 
blood-forming organs as well as upon the gastrointestinal tract and the elimi- 
native organs. While the anemia shows no especial characteristics as regards 
the number of red cells and the amount of hemoglobin, yet the peculiar granu- 
lar degeneration and the polychromatrophilia are sufficient to differentiate 
this type from most of the other secondary anemias. The basophilic degen- 



596 DIAGNOSTIC METHODS 

erations of the reds is more marked in lead anemia than in almost any other 
condition and usually runs parallel to the severity of the clinical symptoms of 
the case. In arsenical poisoning also we occasionally find a slight amount of 
granular degeneration of the red cells, but the hemoglobinemia in this latter 
condition will differentiate it from lead anemia. 
(B) Leukemia. 

Although acute forms occur, leukemia may be regarded as an essentially 
chronic condition which is characterized on the one hand by definite changes 
in the lymphatic and myeloid tissues of the body, and, on the other hand, by 
certain peculiar changes in the number and relations of the various cellular 
constituents of the blood. These latter conditions must be regarded as purely 
symptomatic of the preceding states and not as the direct pathological condi- 
tion in themselves.^ Leukemia has been classed as a primary anemia, al- 
though the changes in the blood are here more directly related to variations 
in the white cells than to characteristic changes in the red corpuscles; yet we 
do find a diminished red count as well as a diminution in the amount of hemo- 
globin in this condition. It is a disease marked by the constant presence in 
the blood of granular mononuclear or polynuclear cells, or an increase of the 
nongranular cells with round nuclei. While the leucocyte count is almost 
invariably increased to a marked extent, we find cases in which the number of 
cells is normal, but we find great deviations from the normal relations of these 
white cells. 

While the tendency is becoming more and more general to regard this 
condition as a single entity, manifested by various blood pictures, yet we find 
the cells grouping themselves together in such definite ways that we are justi- 
fied in dividing leukemia into three general types, with transitions from one 
to the other form.^ These types are (i) splenomyelogenous leukemia or 
"myelemia," (2) lymphatic leukemia or "lymphemia," (3) mixed leukemia. 
Each of these types shows a distinct blood picture which permits of the classi- 
fication of the condition studied. 

(i) Splenomyelogenous Leukemia. 

This condition was formerly subdivided into two distinct types — the true 
splenic and the myeloid leukemia. However, practically none of the cases 
reported could be definitely classed under either one of these headings, as the 
blood picture was always referable to disturbance in both the spleen and 
marrow. This type of leukemia is characterized by a marked increase in all 
of the granular cells, especially of the neutrophile, eosinophile, and basophile 
types, while the nongranular cells are not so characteristically increased. 

^ It is probable that leukemia, both of the chronic and acute types, is of infectious 
origin. Streptococci and the bacillus hodgkini have both been isolated from these cases. 
See Steele, Boston Med. and Surg. Jour., 1914, CLXX, 123; Simon and Judd, Jour. Am. 
Med. Assn., 1915, LXIV, 1630; Wilbur, Ibid., 1915, LXV, 1255; Dias, Brazil-Med., 1915, 
XXIX, 305, and 329; Ward, Brit. Jour. Child. Dis., 1917, XIV, 10. 

2 It is probably true that a classification of these types on purely morphological bases is 
futile. See von Bombard, Ztschr. f. klin. Med., i9i4,LXXX, 506; Emden and Rothschild, 
Deutsch. Arch. f. klin. Med., 1914, CXV, 304; Panton, Tidy and Pearson, Quart. Jour. Med., 
1914, VII, 304; Rummo, Rif. Med., 1914, XXX, 897 and 926; Castellino, Ferrata and 
Rummo, Gazz. d. osp., 1914, XXXV, 1813; Rotky, Zentralbl. f. inn. Med., 1914, XXXV, 
953; Martelli, Rif. Med., 1914, XXX, 1233, 1271, 1294 and 1324. 



PLATE XXVI. 




Blood in Spleno-myelogenous Leukemia. (Tri-acid Stain.) 



THE BLOOD 597 

Gross Appearance. 

The gross appearance of the blood is normal even though the leucocytes 
are increased to an enormous extent. In extreme cases it may appear pale 
and opaque and does not flow from a puncture as readily as normal blood. 
In making smears of such blood, the preparations appear granular and are not 
readily spread so that the future examination is rendered somewhat difficult. 

Red Cells. 

As a rule, these cells are diminished in number, but the oligocythemia is 
of a mild degree, the average count being about 3,000,000 although it may run 
as low as 1,500,000. As a general rule, the red cells diminish in proportion to 
the increase in the number of white cells. Occasionally we find cases in which 
an oligocythemia persists with a normal or slightly increased leucocyte count. 
Such a condition might lead to the diagnosis of pernicious anemia, unless the 
differential count was carefully studied. The red cells are usually pale and 
of the chlorotic variety. Very little degeneration of the red cells is observed, 
microcytes and macrocytes are rare, but a few poikilocytes are seen in prac- 
tically all cases. Polychromatophilia is more or less common and cells 
showing basophilic granulations appear with more or less frequency. Normo- 
blasts are very common in this condition, yet their absence does not rule out 
the diagnosis of leukemia. Megaloblasts and gigantoblasts are frequently 
seen and are sometimes many in number, although they rarely if ever exceed 
the normoblasts in number. 

Hemoglobin. ' 

The hemoglobin is reduced to a somewhat greater extent than is the num- 
ber of red cells, the color index being about 0.6 and the average percentage of 
hemoglobin about 40. 

Leucocytes. 

In this condition we find the leucocytes increased, as a rule, to a very 
marked degree, counts running as high as 750,000 having been seen by the 
writer. Osier gives as his average for the white count 298,700, while the 
average may vary to a slight extent at different periods of the day. Some of 
the cases show a uniformly high count, others a moderate count and a few 
others a low count of about 100,000 cells. It is this increase in the number 
of white cells which gives the blood its peculiar opacity in this condition and 
may make a diagnosis possible by mere inspection. In some cases of leuke- 
mia we find a normal count of white cells while in others the count may be 
similar to that of a simple leucocytosis. It is the differential count in com- 
bination with the large increase in cells which should be considered charac- 
teristic, rather than a simple increase in itself. 

Differential Count. 
Neutrophile Myelocytes. 

These cells are large mononuclear cells with neutrophile granules. They 
are present in large numbers, averaging about 35 per cent, of all the leucocytes 
present. A diagnosis of leukemia is almost always possible when we have 



59^ DIAGNOSTIC METHODS 

such an extreme neutrophile myelocytosis along with an extreme leucocyto- 
sis. These myelocytes appear in two forms: (i) the large myelocytes of 
Cor nil, which may be as large as 30 microns in diameter and have a large, 
pale, eccentric nucleus which is poor in chromatin. These cells are seen 
only in splenomyelogenous leukemia and in some of the secondary leukemias 
of children and must be regarded as practically pathognomonic of this con- 
dition.^ (2) Small myelocytes about the size of the normal polynuclear 
leucocytes with a centric, round nucleus staining deeply with the various 
aniline dyes. We find all gradations between these large and small mye- 
locytes, sometimes observing a few which are about the size of the red cell. 
The granulations of these cells are sometimes numerous, but may be 
entirely lacking so that they may be indistinguishable from the large 
lymphocytes unless the pale quality of their nuclei is remembered. The 
degenerative changes in these myelocytes are few in number and are limited, 
as a rule, to the hydropic form usually seen in chlorosis. 

Polynuclear Neutrophile Leucocytes. 

These cells are relatively diminished, their average, according to Cabot, 
being about 46 per cent., although an absolute increase is present, amounting 
to as much as 60,000 to 75,000 cells. Marked variations in the size of these 
cells are common, some of them being very large, some very small and no defi- 
nite relation existing between the numbers of the large and small cells. These 
variations in size of the polymorphonuclear cells are rarely if ever seen in 
ordinary leucocytosis. It is very common to find cells with irregularly 
shaped nuclei and with more than one form of granule, which may vary in 
tint depending on the method of fixation. Marked degenerative changes in 
these cells are very common. Thus we find their stickiness is increased, 
their nuclei usually pale and frequently showing karyokinetic figures. All 
grades of variations in the granulations may be observed, the granular 
cytoplasm being occasionally replaced by a homogeneous highly refractive 
material. 

Eosinophiles. 

The eosinophile cells are usually much increased in the splenomyeloge- 
nous form of leukemia, but their percentage relations to the other leucocytic 
forms are practically normal. Their number may run from 3,000 to 100,000 
the average absolute number being about 12,000, while their percentage is 
about five. The total number of these cells per cmm. greatly exceeds that 
found in any other condition so that we accept, with Ehrlich, such an increase 
as pathognomonic of splenomyelogenous leukemia. These cells appear in all 
modifications, some of them being very small, while some of them are very 
large. The eosinophile form of myelocyte occurs in large numbers, but never 
is as numerous as is the neutrophile myelocyte. We occasionally observe all 
forms of transition between the myelocyte and the eosinophile leucocyte, the 
eosinophile myelocytes occasionally forming the majority of the eosinophile 
cells. The granulations of these cells may be of uniform size and staining 

^ See Klein, Deutsch. med. Wchnschr., 1913, XXXIX, 2513. 



THE BLOOD 59^ 

quality or there may be some basophile granulations among the eosinophils 
while the granulations themselves may vary greatly in size. Ewing considers 
eosinophile myelocytes with granules of unequal size and density of stain as. 
pathognomonic of myelocythemia. 

Basophiles. 

According to Ehrlich, we always find an increase in the number of mast 
cells in leukemia, there absolute increase being in some cases greater than 
that of the eosinophiles and is always proportionately higher. This in- 
crease is so marked as to constitute a very reliable diagnostic feature of 
the blood. The number of basophiles may run as high as 140,000 (Taylor),, 
while the percentage may vary from 5 to 47. 

Lymphocytes. 

The number and proportions of lymphocytes in the splenomyelogenous. 
leukemia vary in different cases and at different times in the same case. As 
a rule, their percentage is reduced averaging about 10, while an absolute in- 
crease is usually present, this increase having no uniform relationship ta 
the stage or character of the disease. These cells vary much in size, the 
large cells usually outnumbering the small ones. Large mononuclear cells 
with very faint cytcreticulum and vesicular nucleus occur in large numbers 
in this form of leukemia and seem to have no special significance, although, 
they may be mistaken for the large lymphocyte or for the myelocyte. De- 
generative changes are observed in both the small and large lymphocytes in 
leukemia; thus the nuclei of the small cells may become incurved and bilobed 
or even trilobed, while the cell body remains basophilic (Rieder) . 

Points in Diagnosis. 

An excessive leucocytosis, with a large proportion of neutrophile mye- 
locytes, the presence of a large number of eosinophile myelocytes and of 
basophile cells, the presence of atypical cells, both of the mononuclear and 
polynuclear variety, and large numbers of nucleated red cells are the chief 
characteristics. Any one of these points may fail for a time, but will usually 
be evident at some stage of the disease. The large size of the myelocyte 
is much more characteristic than the mere presence of these cells, so that 
we should confine our diagnosis to cases which show irregularity in size, stain- 
ing qualities, and degenerative reactions of these cells rather than to those 
showing merely an increase. The presence of the large number of eosino- 
philes, especially those showing granules of irregular size and staining quali- 
ties, is a very important point to be remembered in the diagnosis of this- 
condition.^ 

(2) Lymphatic Leukemia (Lymphemia). 

In this form of leukemia we observe a marked increase in the number 
of mononuclear nongranular cells in distinction from the previous form of 

1 Seepieballa andjEntz., Foiia Haemat., 1913, XV, 59; also, Ghon and Roman, Ibid., 72; 
and Milne, Jour. Am. Med. Assn., 1913, LX, 822. Goodall (Boston Med. and Surg. Jour., 
1914, CLXX, 789) discusses the nitrogenous metabolism in this condition; Tiberti, Speri- 
mentale, 1918, LXXII, 482; Sweet, Southwest. Med., 1919, III, 19. 



6oo ' DIAGNOSTIC METHODS 

leukemia in which the increase is rather in the number of the granular types. 
While a variety of the mononuclear nongranular cells are present, there is 
usually observed a predominance of one particular form and size, in some 
cases the small mononuclear cell with a narrow ragged rim of protoplasm, in 
others the cells of the large lymphocyte type, and in others large cells whose 
protoplasm is basophilic or in some cases distinctly acidophilic.^ 
Red Blood-cells. 

In this form of leukemia we find a much greater anemia than in the 
splenomyelogenous form, although we may observe a normal red count for 
some time. The number of cells varies between 1,500,000 and 4,000,000, 
while the average percentage of hemoglobin is about 37 per cent. Nucleated 
red cells are rare in this condition, yet in the severer cases we may find them as 
numerous as in the splenomyelogenous type. All forms of degeneration noted 
under the previous type of leukemia are occasionally seen in this latter form. 
Leucocytes. 

The leucocytes are, as a rule, increased, the average being about 145,000, 
according to Osier. In this form we may find aleukemic periods which may 
last for a considerable period of time, the count usually rising just before death. 
Differential Count. 

According to Grawitz the cases of lymphatic leukemia may be divided 
into (i) those in which the increase of leucocytes is especially in the small 
mononuclear variety, (2) those showing an increase in the medium-sized 
cells with basophilic homogeneous protoplasm, and (3) those in which the 
cells which predominate are very large and usually degenerated. All these 
forms may occur together and may vary in the same case at different times. 
These mononuclear cells may constitute as high as 99 per cent. (Osier) of the 
total number of leucocytes. These leucocytes show in a very large number 
of cases much degeneration either of the protoplasm or of the nucleus, very 
few of the cells showing mitosis which is so common in the splenomyelogenous 
form. In this type of leukemia polymorphonuclear cells are rare, eosino- 
philes usually absent, and myelocytes and basophiles rarely if ever present. 
This type of leukemia is not easily amenable to diagnosis, especially in differ- 
entiating it from some cases of sarcoma in which the blood may show a simi- 
lar picture. 

In some cases we find a lymphatic leukemia with a considerable number 
of myelocytes both of the eosinophile and neutrophile type. This has led 
to the differentiation of a ''mixed leukemia," which does not seem to be ad- 
visable as we may find myelocytes in the pure lymphatic type of this disease. 

(3) Acute Leukemia. 

This form of leukemia is characterized by its brief course (from six to 
eight wxeks), by the severity of its symptoms, the frequency of the hemor- 
rhagic diathesis, rapidly developing cachexia, and death. This condition 

^ See Xakamura, Deutsch. Ztschr. f. Chir., 1914, CXXXII, 275; Jackson and Smith, 
Boston Med. and Surg. Jour., igi.'^, CLXXII, 136; Fleischmann, Folia Haem., 1915 XX, 
17; Suter, Cor. Bl. f. Schweiz. Aerzte, 1915, XLV, 1281; King, Bull. Johns. Hopk. Hosp., 
1917, XXVIII, 114; Packard and Ottenberg, Jour. A. M. A., 1917, LXVIII, 954. 



PLATE XXVII. 




Lymphatic Leukemia. (Tri-acid Stain.) 



H 



THE BLOOD 6oi 

occurs chiefly in young people and is usually of the lymphatic type,^ although 
a few cases of the .myelogenous variety have been reported (Billings and 
Capps).^ In all cases the anemia is extreme, the red cells usually running 
below 1,000,000 and the hemoglobin as low as 10 per cent. There is no 
type of cell which is characteristic of this form, although the cells are much 
more uniform in size than in the chronic states of this disease. In some 
cases nearly all of the cells have a basophile protoplasm, while in others 
they show acidophilic properties. Nucleated reds are usually rare, although 
they may be present in fairly large numbers. The drop in the count of red 
cells is usually sudden and denotes rapid blood destruction. The leucocyte 
picture resembles closely that of acute infections. 

(C) Pseudoleukemia. 

Under the heading Pseudoleukemia have been grouped a great variety of 
diseases, which have in some cases the external appearances of the disease, 
such as the glandular swelling, splenic tumor and progressive cachexia without, 
however, showing the blood picture which is so characteristic of leukemia. 
On the other hand, we find conditions which have little in common with the 
clinical findings of leukemia and yet show a blood picture similar in some 
respects to that of leukemia. It is difficult to group all of these cases under 
one heading, as the blood picture is not characteristic for any one of these 
conditions. 

(i) Hodgkin's Disease. 

This condition, first described by Hodgkin in 1823, is characterized by 
chronically progressing cachexia with enlargement of the lymph-glands and 
spleen. It has been called, synonymously, lymphatic pseudoleukemia, lym- 
phosarcoma, malignant lymphoma, lymphatic anemia and aleukemic mye- 
losis.^ It is probably infectious in nature,- the etiologic factor being probably 
the Bacillus hodgkini (see Chap. XI). The blood characteristics in this 
disease are more particularly those of a true cachexia, the red ceUs showing 
a diminution in the number which may be as low as 2,200,000, but which 
is usually between 3,000,000 and 4,000,000. The more severe and pro- 
nounced the signs of anemia and cachexia the lower the number of cells, the 
number in these cases running as low as 1,500,000. Morphologically, the 
red cells show much less deviation from the normal than in other severe 
anemias, the average size of the cell being usually normal, degenerations of 
the red cells appearing only in the very severe conditions, microcytes and 

1 See Strauch (Am. Jour. Dis. Child., 1913, V, 43) for a discussion of this condition in 
children. Also, Beltz, Deutsch. Arch. f. klin. Med., 1913, CXIII, 116; Ballagi, New York 
Med. Jour., 1914, XCIX, 68; Yamakawa, Mitt. a. d. Med. Fak. Tokyo, 1914, XI, 115; 
Citron, Berl. klin. Wchnschr., 1914, LI, 332; Deutsch. med. Wchnschr., 1914, XL, 629; 
Brinchmann, Norsk Mag. f. Laegevid., 1915, LXXVI, 1473; Stein, Med. Record, 1916, 
XC, 147; Gasbarrini, Policlinico, 1919, XXVI, 996. 

2 See Beifeld, Arch. Diag., 1915, VIII, 244; Marshall, Arch. Int. Med., 1915, XVI, 
1045; Sappington, Am. Jour. Med. Sc, 1916, CLII, 238; Knox, Am. Jour. Dis. Child., 
1916, XI, 462; Ross, Lancet, 1916, II, 940; Simon and Rosenthal, Jour. A. M. A., 191 7, 
LXIX, 2168; Theo., Jour. Med. Res., 1918, XXXVIII, 385. 

3 See Hirschfeld, Ztschr. f. klin. Med., 1914, LXXX, 126; Ghedini, Gazz. d. osp., 1914, 
XXXV, 2037; Ibid., 1915, XXXVI, i, 33, and 593; Ragusin, Sem. Med., 1915, XXI, 57; 
Chosrojeff, Folio Haem., 1915, XX, ^y, Block. Ugesk. f. Laeger, 1916, LXXVIII, 831. 



6o2 DIAGNOSTIC METHODS 

macrocytes, as well as nucleated erythrocytes, being very unusual except in 
the late stages. The hemoglobin content runs parallel to the number of red 
cells, being the lowest in those cases showing very low counts. The leuco- 
cytes are slightly increased, averaging about 12,000. This failure of a leuco- 
cythemia enables us to differentiate Hodgkin's disease from a true lymphemia. 
The differential count of the leucocytes may show a relative lymphocytosis, 
the relation of the lymphocytes to the polynuclear cells being as three to one 
instead of the normal one to three. ^ Ehrlich and Pinkus consider this relative 
increase of lymphocytes characteristic of true pseudoleukemia in contradis- 
tinction to sarcomatous and other lymphomatous conditions. According 
to Grawitz, an increase in the leucocytes is associated with an unsatisfactory 
course of the disease, while a diminution in the number is observed as the 
disease progresses toward convalescence. 

Cases have been reported which would seem to point to the transition 
of Hodgkin's disease into a true leukemia, so that we may find irregular prog- 
ress of a pseudoleukemia as an evidence of a transitional stage. 

(2) Tuberculosis of the Lymph-glands. 

Why this condition has been classed as a pseudoleukemia is questionable, 
as the blood picture shows nothing beyond a secondary anemia with cachexia 
or may show even a normal red and white count. A large increase in the 
number of leucocytes, which is so characteristic of leukemia, is rarely seen, 
although a true leukemia may arise in the course of a glandular tuberculosis. 
The differentiation of this condition should be based upon examination of 
an excised gland, which will show distinct tuberculous lesions and usually 
will contain the tubercle bacilli in demonstrable numbers. ^ In other cases 
we may find a simple lymphoid hyperplasia without any distinct inflam- 
matory changes and without demonstrable bacilli. Such nodes should be 
tested by inoculation experiments as advised by Ewing. Heredity plays 
a great role in the diagnosis of these tubercular conditions, while a scrofulous 
child should always be looked upon with suspicion. A splenic tumor appear- 
ing coincidently with the glandular swelling would speak rather against 
tuberculosis and in favor of a lymphatic pseudoleukemia. The diagnosis of 
this condition by examination of the blood alone is a practical impossibility. 
It is this type of case that is especially amenable to diagnosis by the use of 
the various tuberculin tests. 

(3) Lymphosarcoma. 

The lymphosarcomata usually run their course either as primary benign 
lymphomata or as the malignant sarcomata. The blood findings in these 
conditions show nothing beyond a slight anemia with nothing characteristic 

^ Bunting (Bull. Johns Hopkins Hosp., 191 1, XXII, 114 and 369) considers an incraese 
in the number of transitional cells together with increase of blood platelets as quite char- 
acteristic of true Hodgkin's disease. See, also, Bunting, Bull. Johns Hopkins Hosp., 1914, 
173 and 177, Yates, Ibid., 180; Cunningham, Am. Jour. Med. Sc, 1915, CL, 868; Mellon, 
Ibid., 1916, CLI, 704; Moore, Jour. Infect. Dis., 1916, XVIII, 569; Woolley, Jour. Lab. 
and Clin. Med., 1917, II, 523. 

2 Resort should be made to the antiformin method, if ordinary staining processes are 
not conclusive. 



THE BLOOD 603 

in the appearance of the white cells. The diagnosis must be based entirely 
on the examination of the excised gland or tumor. ^ 

(4) Gummatous Lymphoma. 

An exact diagnosis of this syphilitic swelling of the lymphatic glands is 
at the present time a matter of more or less difficulty. The previous history, 
as well as other manifestations of syphilis, must be studied and a careful 
search made for the presence of the spirochaeta pallida. It is to be said that 
these organisms have been frequently reported in lymphatic enlargements 
which seem to have no direct relationship to purely syphilitic conditions. The 
interpretation of one's findings is of the utmost import as artefacts, which 
commonly appear in preparations of broken-down glandular tissue, resemble 
very closely the spirochaete. The blood condition shows nothing character- 
istic and can have only incidental diagnostic importance. Application of the 
Wassermann serum test might throw much light on the diagnosis. 

(II) General Pathology. 
(a) Blood Changes Following Surgical Intervention. 

Under this heading the writer will not attempt to take up all of the surgical 
conditions, as the vast majority are not associated with any direct hemato- 
logical characteristics. 

As a rule, it may be said that pus formation anywhere in the system will 
cause a leucocytosis. The degree of this leucocytosis averages about twice 
the normal standard, but may greatly exceed this figure in individual cases. 
It must be said, however, that trivial as well as extensive pus formations may 
be accompanied by normal or even subnormal values for the leucocytes. 
This is due to the facts that small pus foci do not cause any systemic reaction 
and extensive pus formation may overcome the power of the system to react 
against the infection. If the pus cavity is well encapsulated, the absorption 
of the toxin from this focus is necessarily limited so that we may find no leuco- 
cytosis, even though a very large pus cavity is present. Thus we find in 
localized peritonitis following appendicitis that a leucocyte count may be 
normal, but may suddenly increase to a marked extent as an indication of the 
rupture of the cavity and an extension of the process. ^ A general rule is 
that a distinct increase in the number of cells in excess of the figure originally 
obtained is indicative of the extension of pus formation and should put the 
surgeon on his guard as to operative interference. 

If the absorption of toxic material from a focus of pus formation is great 
enough to produce a systemic effect upon a patient as manifested by a high 
leucocyte count, we find an anemia characterized by a marked diminution in 
the hemoglobin and the number of red cells, which is parallel in intensity to 
the severity of the poisoning. 

Cases are frequently found in which a low leucocyte count prevails, al- 
though clinical evidence of severe sepsis is at hand. In these cases a differ- 

^See Oliver, Jour. Med. Research, 1913, XXIX; 191; Steiger, Berl. klin. Wchnschr., 
1913, L, 2129; Glanzmann, Deutsch. Arch. f. klin. Med., 1915, CXVIII, 52; Warfield and 
Kristjanson, Am. Jour. Med. Sc, 1916, CLII, 222. 

2 See Friedman, Am. Jour. Med. Sc, 1914, CXLVIII, 540. 



6o4 DIAGNOSTIC METHODS 

ential count of the leucocytes should be made in all cases, as the low leucocyte 
count may throw one off his guard unless this precaution be taken. Should 
the polynuclear neutrophils form 80 to 95 per cent, of the total leucocytes, 
a severe infection is indicated, even though the leucocyte count may be 
subnormal. 

If a leucocyte count does not diminish or even return to normal within 
one to two days after operation, this should be taken as evidence that a rein- 
fection has occurred or that the pus cavity has not been properly drained. 
Recourse must, therefore, be had to measures to overcome the secondary 
infection. 

It has been found that administration of ether and chloroform causes a 
leucocytosis which usually lasts from 24 hours to 48 hours and which may 
interfere with the interpretation of a blood examination. This point must be 
borne in mind in the examination of blood of cases which have shown high 
leucocyte values prior to operative procedure. The differential count in such 
cases, however, will show only slight variations in the proportions of the differ- 
ent types of cells so that one may judge as to the cause of the secondary 
leucocytosis by determination of the polynuclear cells. The number of red 
cells as also the amount of hemoglobin are very markedly reduced in some 
cases following the administration of an anesthetic, so that a direct secondary 
anemia may be the result. This fact has led to the refusal by many surgeons 
to resort to operative procedure in cases which show as low as 30 per cent, 
hemoglobin prior to operation. ^ 

As the blood changes in surgical conditions, which are not accompanied 
by pus formation, are not especially characteristic and are more especially 
associated with the diseases of the special organs, the writer will refer such 
discussions to other headings. 

(b) Blood in Constitutional Diseases. 
(i) Diabetes Mellitus. 

In diabetes mellitus the changes in the cellular content of the blood are 
not very marked. The leucocytes may be subnormal, normal, or slightly in- 
creased, usually a very slight leucocytosis being observed. The amount of 
hemoglobin is usually reduced to a very sHght extent, while the number of 
red cells may be slightly increased. 

One of the most striking peculiarities of the blood in diabetes is the presence 
of an abscess of fat (lipemia). Microscopic examination usually reveals the 
presence of the extracellular globules, but in some cases fat is present in 
sufficient amount to permit of demonstration by macroscopic methods. 
Glycogen has been found both in the plasma and in the leucocytes of diabetic 
blood and shows the peculiar characteristics discussed in the section on 
lodophilia (p. 563). Certain peculiarities of the blood in diabetes have led 
to the introduction of tests supposed to be characteristic for such blood. 
These tests are occasionally of diagnostic value, although a diagnosis may, as 
a rule, be made even when these tests do or do not obtain. 

Bremer's Test.— This test is based upon the fact that diabetic blood does 

^ See Cullen, Surg., Gynec. and Obst., 1913, XVII, 276. 



THE BLOOD 605. 

not stain to any appreciable extent when treated with certain aniline dyes. 
Thick smears of the blood are made upon slides and are fixed by dry heat. 
These smears are then covered with a i per cent, aqueous solution of Congo 
red and allowed to stain for a few minutes, after which they are rinsed in 
water and dried. Diabetic blood will be stained either a faint yellow or not 
at all, while normal blood will be colored a bright red. A i per cent, solution 
of Biebrich scarlet will stain the diabetic blood intensely while the normal 
blood is unstained. Bremer's original staining solution was made up as 
follows: Saturated watery solution of eosin and of methylene blue are mixed 
in equal proportion when a precipitate forms which is filtered, washed, dried 
and powdered. To 24 parts of this powder are added six of powdered methyl- 
ene blue and one of eosin. One-twentieth of a gm. of this mixed powder is 
dissolved in 10 c.c. of SS per cent, alcohol and forms the staining solution in 
which the specimens are stained for four minutes. The diabetic blood stained 
by this solution has a greenish tint, while normal blood is reddish violet. 

Similar reactions have been found in normal blood, in leukemia, in exoph- 
thalmic goiter, in Hodgkin's disease, in multiple neuritis, and in some cachec- 
tic conditions, but the reaction in all these cases is very inconstant. Accord- 
ing to Bremer, cases of renal diabetes do not give this reaction, which is more 
characteristic of the pancreatic type of the disease. 

Williamson's Test. — This test is performed as follows: Two drops of 
blood (20 cmm.) are dissolved in four drops (40 cmm.) of water and to the 
solution is added i c.c. of a i to 6,000 aqueous methylene blue solution. To 
this is added four drops (40 cmm.) of 6 per cent, solution of liquor potassae 
and the test-tube placed in boiling water for four minutes. Diabetic blood 
will decolorize the solution, while normal blood leaves it a deep blue. The 
same effect is observed by using diabetic urine instead of the blood. 

(2) Gout. 

Little information is available as to the variations in the number of the red 
and white cells in the gouty condition. The recorded observations show that 
acute gout has little effect upon the number of red cells and upon the amount 
of hemoglobin, while chronic gout may be accompanied by an anemia which is 
more directly referable to causes other than the gouty condition itself. The 
leucocytes are usually increased in the acute attack, while in the chronic form 
the leucocytosis is of a more moderate grade. Neusser in working upon the 
blood of gouty patients found many polymorphonuclear leucocytes whose 
nuclei were surrounded by basophile granules — the so-called perinuclear baso- 
phile granules. These he considered diagnostic of the uric acid diathesis, but 
Futcher and Simon have found them in many other conditions, while Ehrlich 
regards them as artefacts. 

While the chemistry of the blood in gout has been the subject of much 
investigation for a long period, nothing of diagnostic importance has been 
found in the chemical properties of the blood. The excess of uric acid 
has been shown not to be pathognomonic of gout, as it is present in many 
other conditions which are cUnically far removed from the gouty state. 



6o6 DIAGNOSTIC METHODS 

(3) Addison's Disease. 

This disease is usually associated with a severe grade of anemia, the nura- 
ber of red cells being reported as low as 1,120,000 while the percentage of 
hemoglobin is coincidently reduced. The leucocytes are usually diminished, 
but may be slightly increased, while the relative proportions of the different 
cells are not markedly changed. As the disease progresses unfavorably, a 
relative lymphocytosis may be observed, but this is not always the case.^ 

(4) Rickets. 

The state of the blood in rickets varies with the extent and severity of the 
primary disease and is markedly affected by complications. Cases are re- 
ported in which the red cells are practically normal and in which the hemo- 
globin was only very slightly reduced. This disease is not associated with any 
special type of anemia, although the hyperemia of the bone-marrow might be 
expected to yield a large number of nucleated red cells. The usual condition 
of the blood in rachitic children is of the type of simple chlorotic anemia. A 
grave secondary anemia is seen in many cases in which there are serious 
complications. The leucocytes in practically all cases of rickets are increased, 
but may not exceed the normal limits for the child. As is usual in the blood of 
a child, the lymphocytes are increased while the eosinophile cells are often 
relatively numerous.- Just exactly what the cause of the leucocytosis in 
rickets is must be left to the realm of hypothesis, as neither the gastroenteritis 
nor the hyperplastic splenitis are sufficient to explain all cases. 

(5) Myxedema. 

In this disease we usually find an anemia of the secondary chlorotic type 
along with a moderate leucocytosis. The number of cells is usually somewhat 
diminished, although their size is usually increased. The proportion of the 
different leucocytes does not vary, although a few myelocytes are sometimes 
seen in the blood, which is numerically normal in other respects. 

Although many studies of the chemistry of the blood in myxedema have 
been made, little knowledge has been forthcoming as to the exact cause of 
this toxemia. It is highly probable that the changed activity of the thyroid 
gland in this disease influences other organs to such an extent that slight 
anemia is the result and we should, therefore, assume that this anemia is more 
a secondary one than a primary result of thyroid insufficiency.^ 

(c) Blood in Acute Infectious Diseases. 

In a study of the blood in acute infectious diseases, we must remember 
that there are certain general rules which apply to all of such diseases with 
a very few exceptions. The interrelation of the fever with the resistance of 
the system in general is so close that it is hard to say in any given case whether 
certain changes in the blood are or are not due to the increased temperature 
in itself. There can be little doubt that a high temperature working over a 

^ See Buettenmiiller, Med. Klin., 1914, X, 1354; Engelsmann, Folia Haemat., 1915, 
XIX, 333; Scheltema, Nederl. Tijdschr. v. Geneesk., 191 5, I, 1767- 

2 See Ostrowski, Folia Haemat., 1912, XIII, 305. 

^See Salvatore (Riforma Med., 1913, XXIX, 1373) for a discussion of the blood 
changes in exophthalmic goiter. 



THE BLOOD 607 

considerable period of time will destroy large numbers of red cells and will 
bring on various changes in the blood which might be misinterpreted. A 
rather extensive concentration of the blood along with a progressive loss of 
albumin is observed in practically all conditions associated with fever. We 
should, therefore, expect to find the number of red cells increased at the out- 
set of such condition, owing to the concentration of the fluid portion, while a 
distinct anemic condition may become evident only after the lapse of some 
time. 

Fever in itself does not have a large influence upon the number of leuco- 
cytes, but it may be stated as a general rule that most infectious diseases 
(the exceptions being malaria, typhoid, tuberculosis, influenza, and measles) 
are associated with an increase in the number of white cells. This is not an 
invariable rule, as will be seen under the discussion of the various infectious 
types. The leucocytosis so commonly seen associated with infection is no 
doubt due to the action of the bacteria themselves and of their products 
upon the leucocytes. The positive chemotaxis which bacteria and their 
toxins exert upon the leucocytes is very marked. Moreover, as the blood 
becomes laden with these abnormal products, new leucocytes are thrown into 
the circulation to aid the old ones in their phagocytic action. Just what sub- 
stances are accountable for the increased opsonic power of the serum in any 
specific infection must be left undecided for the present. 

In the following discussion of the various infectious diseases, the writer 
will not attempt to give more than a brief discussion of the blood changes in 
these separate conditions, leaving associated questions to other writers. 

(i) Pneumonia. 

This disease is, hematologically, one of the most definitely characterized 
of all the infectious diseases. While the physical findings of this condition 
are largely local, the systemic effects are so marked that definite changes are 
seen in the blood both in the early and in the later stages of the infection. 
While showing so many characteristic findings in the blood, it is, at the same 
time, one of the most obscure in its relations to opsonins and to phagocytosis. 
Just why the virulent pneumococci should be so little capable of phagocy- 
tosis and why the addition of attenuated cultures of pneumococci or of ex- 
tracts of virulent organisms should increase this phagocytic power of the 
leucocytes is at present very uncertain. We must, therefore, leave the dis- 
cussion of this phase of pneumonia as well as of other infectious diseases to the 
section on Bacteriology of the Blood. 

In pneumonia we find in the early stages that the blood is somewhat con- 
centrated owing to the action of the increased temperature, while this concen- 
tration gradually increases as the exudate forms. Such a condition can lead 
only to an increased count of both the red and the white cells. As the dis- 
ease progresses, the number of red cells shows a slight but a steady decline, 
which points not only to a destruction of the red cells, but to a diminished 
formation. This decrease in the number of red cells is occasionally seen only 
at the time of crisis, while in the cases in which the diminution is gradual the 



6o8 DIAGNOSTIC METHODS 

period of diminution does not usually exceed ten days. It will be seen, there- 
fore, that the red cells in pneumonia may be about normal in number and at 
the same time an anemia may be present which becomes evident only after 
the disease has progressed for some time. The red cells are, as a rule, nor- 
mal in appearance, but an occasional polychromatophilic cell may be seen, 
especially in the severe cases. Rarely normoblasts may be observed and very 
rarely megaloblasts. 

The hemoglobin usually shows a greater reduction than does the number 
of red cells, which decrease may become evident only after the fever has sub- 
sided. A reduction in the hemoglobin below 60 per cent, is very unusual in 
pneumonia of the pure type. 

Pneumonia is one condition in which the leucocyte count may prove of 
great value. A leucocytosis appears in most of the cases, being absent in 
very mild cases as well as in those very severe ones which show very feeble 
resistance of the organism toward invasion by the pneumococci.^ Rieder's 
observations are very interesting on this subject. He has found that the 
leucocytosis of pneumonia is more a function of the intensity of the infection 
and the degree of resistance toward this infection than it is of the fever or of the 
extent of the exudate. ^ A leucocytosis which may reach 12,000 to 20,000 
appears very early in the course of this disease, and is usually evident at the 
time of the chill or immediately following. A steady increase is sometimes 
observed in the number of the white cells so that the maximum is usually 
reached just before the crisis. It is to be said that rapid extension of the dis- 
ease as well as continuous high temperature may cause much irregularity in 
the count, cases being reported in which the leucocytes are high at first and 
steadily diminish as the patients grow worse. Others may show a sudden in- 
crease in the number of ceUs as the time of crisis is approached.^ According 
to Ewing, when the leucocytes increase slowly they usually diminish slowly 
and the disease defer vesces by lysis. The degree of leucocytosis in pneu- 
monia may reach any stage between the normal figure and that of 115,000 as 
reported by Laehr. 

The increase in the number of white cells in pneumonia is largely referable 
to increase in the number of the polynuclear neutrophiles, these cells constitu- 
ting as high as 97 per cent, of the total number of white cells. Associated with 
this polynuclear leucocytosis we have a marked diminution of lymphocytes, 
while the large mononuclear cells usually persist in considerable numbers. 
The eosinophile cells are always much reduced at the height of the leucocyto- 
sis, so that we may not be able to find a single one after very prolonged search. 
Cabot has reported a case in which the lymphocytes constituted 66 per cent, 
of a total of 94,600 white cells, but such a finding is not the usual one follow- 
ing infection with the pneumococcus. As defervescence goes on the polynu- 
clear cells diminish very rapidly, while the lymphocytes increase and the 
large mononuclear leucocytes become very numerous, reaching as high as 

^ See Hess, Am. Jour. Dis. Child., 19 14, VII, i. 

2 See Kline and Winternitz, Jour. Exper. Med., 1913, XVIII, 50. 

3 Dick (Jour. Infect. Dis., 191 2, X, 383) shows that proteolytic ferments develop in the 
blood about the time of crisis. 



THE BLOOD 609 

16 per cent, in a case reported by Tiirk. The eosinophile cells usually appear 
about the time of crisis, but occasionally their appearance is postcritical. 
The degenerative changes seen in the leucocytes in pneumonia are in no way 
different from those observed in other infectious diseases. 

It will be noted, from the above remarks, that the blood changes in pneu- 
monia are those of a mild anemia associated with a high-grade polynuclear 
leucocytosis and a distinct lymphopenia. Too much reliance must not be 
placed on the blood finding in a case of pneumonia, owing to the fact that 
many abnormal cases are present and show results different from the above 
which can be interpreted only by a complete study of the complications in any 
special case. As a general rule, it is to be said that an absence of leucocytosis 
is strong negative evidence against pneumonia, while leucocytosis may serve 
to differentiate this condition from typhoid fever and malaria with which it 
might be confounded, especially where the systemic and cerebral symptoms are 
more pronounced than are the local pulmonary changes. 

(2) Typhoid Fever. 

This condition, like the preceding, is very often associated with such 
marked systemic disturbance that the local intestinal manifestations are 
obscured and the diagnosis rendered somewhat difficult. While typhoid fever 
is a purely infectious condition and subject to the ordinary laws governing 
such cases, yet we find for some reason that the invasion of the blood by 
the specific organism is not associated with a leucocytosis, although the febrile 
rise may be very marked. The study of the characteristic serum reaction for 
typhoid fever as well as of the bacteriology of the blood must be left for a later 
section, the discussion here being limited to the changes in the microscopic 
appearances of the blood. 

In this condition we find the total volume of blood very much diminished 
in the early stages, both as a result of the high temperature and the diarrhea 
and repeated hemorrhages which may occur at any stage of the disease. 
This concentration of the blood leads to an initial polycythemia which may 
last for two or even three weeks. However the characteristic change in the 
red cells is One of a slight and gradual decline, the number of these cells not 
usually falling below 4,000,000. One must be on his guard in an examination 
of the blood in any infectious disease lest he conclude from a slightly increased 
count that no anemia is present. It is a very safe precaution, although rarely 
followed, to determine the specific gravity of the blood so that one may com- 
pute the degree of concentration. In this way he may be able to show that 
the number of cells normally present in such a concentrated blood is much 
higher than in the case of suspected typhoid fever which he is examining. At 
any rate, it is wise to make frequent determinations of both the number of 
cells and of the hemoglobin, as a reduction in both of these elements takes 
place gradually as in the cases reported by Thayer and Da Costa. 

The reduction in the amount of hemoglobin is in some cases very marked, 
being as low as 50 per cent, in one case observed by the writer. The repeated 
hemorrhages which so often occur in typhoid fever may cause marked varia- 



6lO DIAGNOSTIC METHODS 

tion in this value. The morphological changes which occur in the red cells 
are not very marked, as a rule, but may be very severe in case much blood is 
lost by frequent hemorrhages. Polychromatophilia is more or less frequent 
and irregularity in the size of the red cells is occasionally seen, while nuclea- 
tion of these cells along with formation of a few megaloblasts may occur in 
severe hemorrhagic cases. 

The leucocytes are usually normal in number in the early stages of uncom- 
plicated cases, but any complication may cause a polynuclear leucocytosis 
which may be confusing to the worker. The behavior of the leucocytes is 
very variable in the early stages, so that one should never rest his diagnosis 
of typhoid fever upon a negative leucocytosis. As the disease progresses, the 
leucocytes show a gradual reduction, especially in the number of the polynu- 
clear cells, which reduction continues until the disease has reached its highest 
point, after which they slowly increase.^ 

The more severe the action of the typhoid toxin the lower is the leucocyte 
count, the reduction not usually going below 2,500 cells, the majority of cases 
showing a count between 4,000 and 6,000. It is not an uncommon thing to 
observe a leucocytosis during the later course of typhoid fever and it is not 
always easy to explain such a condition. Marked hemorrhage, cold baths, 
severe diarrhea, and usually perforation may account for the increase in the 
number of cells, but we do not always find a leucocytosis after such conditions. 

In typhoid fever we find quite characteristic changes in the relations of 
the various types of leucocytes. During the first week the neutrophile cells 
do not, as a rule, increase, while the lymphocytes, especially of the medium- 
size variety, show a progressive rise. The lymphocytes are rarely below 25 
per cent, of the total number of cells and may reach as high as 65 to 70 per 
cent. The eosinophile cells are usually low in number during the febrile 
period, but reappear about the time of defervescence. 

It will thus be seen that the characteristic changes in the blood of typhoid 
fever are a slight anemia, together with a leucopenia and a relative, and in 
some cases absolute, lymphocytosis. Such characteristics are the usual ones 
of typhoid fever, but it is to be remembered that suppurative processes do not 
always produce a leucocytosis, nor is a leucopenia always present in typhoid 
fever. 

(3) Scarlet Fever. 2 

In this condition we find the usual effects of fever manifested in a slight 
concentration of the blood, leading in the early stages to a polycythemia. 
The usual change, however, in the red cells is one of gradual reduction in 
numbers to as low as 3,000,000 cells and occasionally much lower. The 
hemoglobin also suffers quite a diminution, so that the anemia may reach 
quite a severe grade. 

The leucocytes in scarlet fever usually increase in number one or two 

1 See Ravenna, Rif. Med., 191 7, XXXIII, 65 and 93. 

2 See Koessler (Jour. Am. Med. Assn., 1912, LIX, 1528) for a discussion of the recent 
advances regarding scarlet fever. Mallory and Medlar, Jour. Med. Res., 1916, XXXIV, 
127. 



THE BLOOD Oil 

days before the appearance of the rash and continue to increase until quite 
a marked leucocytosis, ranging from 10,000 to 50,000 cells, becomes evident 
at the time of the complete eruption. The degree of the leucocytosis cor- 
responds as a rule with the severity of the disease and in some cases is dimin- 
ished at the time of the eruption, but usually continues for several days and 
may even extend for weeks after the temperature has subsided. 

The increase in the number of leucocytes is largely referable to the poly- 
nuclear cells, these constituting from 85 to 99 per cent, of the total number. 
The lymphocytes diminish in the early stages of the disease, but later rise to 
normal or slightly above normal figures. The eosinophile cells are usually 
normal or even subnormal at first, but steadily increase as the disease pro- 
gresses and reach a degree of 10 to 20 per cent, in the second or third weeks, 
after which they slowly decline. These rules are not invariable in scarlet 
fever, but a severe leucocytosis appearing prior to the period of eruption of 
an infectious fever is practically always suggestive of this disease. In 
some cases the polynuclear cells diminish about the end of the first week and 
the lymphocytes and eosinophiles rapidly increase, leading to a later sec- 
ondary leucocytosis.^ 

(4) Measles. 

This condition shows in itself nothing particularly characteristic in the 
blood, but the absence of definite findings is of great importance in its differ- 
entiation from scarlet fever with which it might be confounded. The red cells 
in this disease are not found to be greatly changed, although a slight reduc- 
tion in their number is usual. A loss of hemoglobin is practically always 
noticed. v 

The leucocytes are usually normal or slightly reduced in number at the 
outset of the disease, being the lowest at the height of the eruption when the 
figure may reach as low as 2,500 cells, returning to the normal within a few 
days after subsidence of fever. A complicating bronchitis may cause a 
moderate leucocytosis of 8,000 to 16,000 cells, but this should not lead one to 
a mistaken diagnosis, as the clinical symptoms of both scarlet fever and of 

1 Dohle (Centralbl. f. Bakteriol., 1911, LXI, 63; Ibid., 1912, LXV, 57; Miinch. med. 
Wchnschr., 191 2, LIX, 1688) has announced the almost constant presence in the polymor- 
phonuclear leucocytes of "inclusion bodies." These are observed near the margin of the 
cell as rod or coccus forms, which stain readily a faint bluish-greert tint with the Giemsa or 
other blood stains. While these bodies are observed in about 95 per cent, of scarlet fever 
cases in the early days of the disease, the number diminishes gradually. They are also 
found in many cases of diphtheria, pneumonia and tuberculosis, yet they are very infrequent 
in cases of serum sickness or of scarlatiniform rashes. Their presence is, therefore, not 
pathognomonic but their absence almost excludes scarlet fever. See Kretschmer, Berl. 
klin, Wchnschr., 1912, XLIX, 499; Deutsch. med. Wchnschr., 1912, XXXVIII, 2163; 
NicoU and Williams, Arch. Pediat., 191 2, XXIV, 350; Ahmed, Berl. klin. Wchnschr., 191 2, 
XLIX, 1232; Kolmer, Am. Jour. Dis. Child., 191 2, IV, i; Granger and Pole, Brit. Jour. 
Child. Dis., 1913, X, 9;Lippmann and Hufschmidt., Zentralbl. f. inn. Med., 1913, XXXIV, 
369; Schippers and de Lange, Berl. klin. Wchnschr., 1913, L, 544; Bongartz, Ibid., 544; 
Schwenke, Miinch. med. Wchnschr., 1913, LX, 752; Pappenheim, Folia Haemat., 1913, 
XV, 379; Cummins, Jour. Med. Research, 1913, XXVII, 529; Nicoll, Arch. Pediat., 1913, 
XXX, 346; and Brinchmann, Berl. klin. Wchnschr., 1913, L, 1248; Massini, Med. EUin., 
1913, IX, 1729; MacEwen, Jour. Path, and BacterioL, 1914, XVIII, 456; Rosanoff, Arch, 
f. Kinderhkde., 1914, LXII, 321; HiU, Boston Med. and Surg. Jour., 1914, CLXX, 792; 
Isenschmid and Schemensky, Miinch. med. Wchnschr., 19 14, LXI, 1997; Kaiser and 
Lowy, Deutsch. Arch. f. klin. Med., 1914, CXVI, 82; Rehder, Ibid., 1914, CXVII, 37. 



6l2 DIAGNOSTIC METHODS 

measles should be well-established at the time of the complicating bronchitis. 
An eruptive fever in the second or third day of its course should be considered 
scarlet fever, or at least scarlatina, if a leucocytosis is present, while if the 
disease be measles the number of leucocytes will be normal or even subnormal 
in the absence of extensive bronchitis.^ 

(5) Variola. 

This condition is associated with more or less extensive destruction of 
the red cells. ^ In the early stages of the disease the red count may be slightly 
above normal, owing to the concentrating effect of the fever, but later the red 
cells will show a sudden reduction. This reduction is especially noticeable 
in the cases associated with extensive pustulation, when the septic process has 
such a marked influence in destroying the red cells. The hemoglobin is 
usually reduced in degree parallel to the diminution in red cells. 

In most of the cases of small-pox we find a distinct leucocytosis which 
may run from 10,000 to 20,000 as a rule, but has reached as high as 41,000 
in the severe cases. This leucocytosis begins with the appearance of the vesicle, 
increases as the exudate becomes purulent, and reaches its height when sup- 
puration becomes extensive; that is, the degree of leucocytosis usually runs 
parallel to the severity of the septic process, the count returning gradually 
to normal as the suppuration subsides. The leucocytosis in small-pox is 
usually of the lymphocytic type, the number of these cells varying from 35 to 
45 per cent, of the white cells. Associated with the increased lymphocyte 
value we find from 5 to 10 per cent, of the large mononuclear leucocytes and 
usually an average of 3 per cent, of neutrophile myelocytes, in some cases 
these latter running as high as 16 per cent. Eosinophils and basophiles 
are occasionally observed, especially in the hemorrhagic form of this 
disease. 

It will thus be seen that small-pox causes a leucocytosis which may reach 
even the degrees given by scarlet fever, but the differential count as show^n by 
the large percentage of lymphocytes in the former and the greatly increased 
number of polynuclear neutrophiles in the latter should make a mistake in 
diagnosis impossible. It is to be remembered that compHcations of true 
abscesses with the pustules of small-pox may increase the percentage of 
polynuclear cells in this disease, but never to such a degree as is shown in 
scarlet fever. 

An examination of the blood of children, who have been vaccinated with 
small-pox virus, shows a distinct leucocytosis of the polynuclear type, reaching 
as high as 20,000 cells. This leucocytosis usually begins on the third or 
fourth day after inoculation and gradually subsides until the end of the period 
of vaccination. 

^ Schwaer (Munch, med. Wchnschr., 1913, LX, 1203) believes that a diminution or dis- 
appearance of eosinophiles at the height of the fever is characteristic of measles. See, also, 
Lucas, Am. Jour. Dis. Child., 1914, VII, 149; and Grumann, Miinch. med. Wchnschr., 
1914, LXI, 132. Hess (Arch. Int. Med., 1914, XIII, 913) discusses the blood picture in 
German measles. 

2 See Proescher (Interstate Med. Jour., 1915, XXII, 427) for a discussion of the cultiva- 
tion of the variola-vaccine virus. 



THE BLOOD 613 

The blood in cases of varicella^ seems to show the same characteristics 
as that of very mild cases of variola, or of vaccinia. The slight leucocytosis 
is usually of the polynuclear type, reaching a degree of 15,000. The large 
mononuclear cells which seem to play such an important role in the differential 
leucocyte picture of variola are for the most part absent in varicella, and 
myelocytes are practically never found in this condition. 

(6) Diphtheria. 

The number of red cells in diphtheria seems to be slightly increased, 
owing to the marked concentration of the blood in this condition. In practi- 
cally all cases of diphtheria the number ranges from a high normal value to 
as high as 7,800,000 reported by Cuffer. As the disease progresses, especially 
after the temperature has fallen, the number of red cells is diminished and 
a coincident decrease in the percentage of hemoglobin is observed. This 
slight anemia is not evident in the early stages of the disease due, no doubt, 
to the abnormal concentration of the blood. 

In this condition, like most infections, we find a leucocytosis ranging 
between 25,000 and 50,000, the higher the leucocytosis the more grave the 
prognosis. In one case reported by Felsenthal the leucocytes numbered 
148,000, but this is very unusual, as the grade of leucocytosis is ordinarily 
proportional to the extent and depth of the membrane. The leucocytosis 
is usually of the polynuclear type, the lymphocytes being also slightly in- 
creased. In some cases a lymphocytosis of 60 per cent. (Ewing) has been 
observed, but this is not the usual finding. The eosinophile cells are reduced 
in number, but are relatively more numerous than in pneumonia. It is in 
diphtheria that we find quite marked degenerative changes in the leucocytes, 
"the leucocyte shadows" and increased acidophile tendency of the neutro- 
phile granules being especially worthy of mention. 

(7) Pertussis. 

The exact work of Barach^ and others has so modified our ideas of the 
changes in the blood in pertussis that I can do no better than to give his 
summary upon these points: 

"In the early stages of this disease there is a leucocytosis with increase 
of all the forms; then a small-cell lymphocytosis becomes conspicuous and 
continues to increase when the other forms have reached their limit. The 
large lymphocytes follow the course of the small ones, but they reach their 
greatest numbers after the small cells have reached theirs. During the stage 
of active lymphocytosis, bilobed, small lymphocytes are frequently seen as 
well as numerous degenerated large lymphocytes, especially the basket forms. 
Then comes the simultaneous falling of the leucocytosis and lymphocytosis, 
while the polynuclears begin to resume their normal proportion. A little 
later the mast cells are observed more frequently, and an occasional myelocyte 

1 See Mensi, Gazz. d. Osp., 1912, XXXIII, 1625; also, Erlenmeyer, Deutsch. med^ 
Wchnschr., 1913, XXXIX, 21; Schatzmann, Cor.-Bl. f. schweiz, Aerzte, i9i3,XLIII, 1515; 
Ztschr. f. klin. Med., 1914, LXXX, 333; Erlenmeyer and Jalkowski, Deutsch. med. Wchn- 
schr., 1914, XL, 646; Force, Jour. Lab. and Clin. Med., 1916, I, 243. 

2 Arch. Int. Med., 1908, I, 602. See, also, Schneider, Miinch. med. Wchnschr., 1914, 
LXI, 303. 



6 14 DIAGNOSTIC METHODS 

may be seen. While the leucocytosis and lymphocytosis continue to fall by 
lysis, an eosinophilia is noted; this continues for a variable time, after which 
the blood formula resumes its normal proportions. During this entire cycle 
the transitionals seem unaffected. 

If we were to speak of the first and second half of the blood cycle in this 
disease, we would say that in the first half the lymphocytes are the prominent 
factors and in the second half the polynuclears and the eosinophiles. 

Qinically, leucocytosis is present at about the time the child first coughs; 
as the coughing goes on, the leucocytosis increases and the lymphocytosis 
becomes very marked. Churchill believes that a lymphocytosis exists in the 
prespasmodic stage and is of extreme importance in early diagnosis. The 
height of the leucocytosis is reached in the spasmodic stage, sometimes early, 
and sometimes in the latter part, the sickest children showing the highest 
grade of leucocytosis. About the time that a marked improvement is noted 
in the child the leucocytosis has decreased, the polynuclears have increased 
and the eosinophilia is present." 

The degree of leucocytosis in this condition varies between 25,000 and 
51,150- 

(8) Acute Rheumatism. 

In this disease we find that the red cells are quite markedly destroyed, 
causing, very frequently, a reduction of 2,500,000 cells. This reduction is 
not always evident in the early stages of the disease, as the blood becomes 
very much concentrated by the marked sweating which is such a prominent 
symptom of the disease. The hemoglobin suffers more than the red cells 
so that we may find the percentage of hemoglobin as low as 60 per cent, in this 
condition. This anemia is one of the characteristic signs of acute rheumatism 
and continues well on into convalescence, the hemoglobin not being as quickly 
restored as are the red cells. A largely increased formation of fibrin has been 
observed and may have some diagnostic importance. 

The leucocytes are increased in proportion to the severity and acuteness 
of the disease, the grade being usually moderate and the type being poly- 
morphonuclear. In the very mild cases we may find no leucocytosis and one 
reaching 20,000 or more is, according to Tlirk, always associated with com- 
plications. As the fever diminishes the leucocytes return to normal and are 
not as much affected by the subsequent attack as by the previous initial one. 
The eosinophile cells are absent only in the early stages, while they are present 
in moderate amounts later in the disease and show a distinct increase after 
defervescence (Loeffier). 

It would be impossible in a work of this character to discuss in detail the 
blood changes in all diseases, whether infectious or non-infectious.^ The 
writer, therefore, has selected under the acute infectious diseases those which 
show the more characteristic changes in the blood and those in which an 
examination of the blood is more frequently called upon to aid in diagnosis. 
Many phases of these diseases have been left to the chapters on bacteriology 
of the blood. 

^ See Barach (Arch. Int. Med., 1913, XII, 751) for blood changes in mumps. Hess and 
Fish (Am, Jour. Dis. Child., 1914, VIII, 385) discuss the blood in infantile scurvy. 



THE BLOOD 615 

(d) Blood in Chronic Infections. 

(i) Tuberculosis. 

The earlier studies of the blood of tubercular patients reveal the fact 
that the blood may show practically no changes which are comparable with 
the pallor of the skin and the degree of the emaciation of the subjects affected. 
An anemia is often seen of the very highest type, but usually one of moderate 
degree is present and even may not exist at all. The degree of anemia is 
independent of the localization of the disease, although pulmonary affections 
are more frequently associated with high-grade anemia than are other tubercu- 
lar conditions. It is to be remembered that pulmonary tuberculosis is so 
frequently associated with extensive hemorrhage that one may not wonder 
at the severe anemia present, yet we find cases in which the regeneration is 
very rapid after severe hemoptysis. As a rule, a mild anemia of the chlorotic 
type prevails; that is, the count is practically normal with the hemoglobin 
somewhat reduced. Occasionally we find a slight lymphocytosis, especially 
of the smaller cells, and only when a secondary infection prevails does the 
leucocytosis take on the polynuclear type.^ The lymphocytosis in tuberculo- 
sis is so common that we usually find even in the sputum an excess of the 
small mononuclear cells in the pure tubercular affection of the lungs. 

In tubercular infection of the meninges we practically always find a 
leucocytosis, but with this exception uncomplicated tuberculosis is not asso- 
ciated with an increase in the number of white cells. It is highly probable 
that the increased percentage of the mononuclear cells is more closely asso- 
ciated with the poor nutrition which the tubercular patient shows than it is 
with any specific effect of the bacillus tuberculosis. Occasionally we find the 
eosinophile cells somewhat increased, especially in pulmonary conditions 
with cavity formation, but it must be remembered that a slight eosinophilia 
will obtain if tuberculin therapy is being used in such cases. 

(2) Syphilis. 

According to Becquerel and Rodier, a moderate grade of anemia is to 
be found in the majority of cases of syphilis, becoming more pronounced 
as the disease progresses. This anemia of syphilis is of the chlorotic type, 
but may increase until the pernicious type becomes established. As a rule, 
the reduction in the number of red cells is moderate, being rarely below 
3,000,000 cells. The hemoglobin is usually relatively more decreased than 
are the cells, and the application of mercury in the treatment of this condition 
frequently lowers this percentage still further, establishing an anemia which 
is directly referable to the mercury. This reduction in the number of red 
cells and in hemoglobin becomes more marked as secondary symptoms ap- 
pear, so that a diagnosis of an initial lesion becomes established by a later 
examination of the blood. 

The increase in the number of leucocytes is largely limited to the second- 
ary and tertiary stages of this disease, as the leucocytes usually remain normal 

^ See Miller and Reid, Arch. Int. Med., 191 2, IX, 609; Miller, Lupton and Brown, Am. 
Jour. Med. Sc, 1912, CXLIII, 683; Ringer, Ibid., 1912, CXLIV, 561; also, Rayevsky, New 
York Med. Jour., 1913, XCVII, 813; Wack, Deutsch. Arch. f. klin. Med., 1914, CXV, 596; 
Morgan, Am. Jour. Dis. Child., 1916, XI, 224. 



6l6 DIAGNOSTIC METHODS 

up to the time of the eruptive stage. The increase in the secondary stage, 
which may reach as high as 20,000, is largely in the number of the small and 
large lymphocytes, but the eosinophile cells may be increased to as high as 
5 per cent, in some cases. In very severe cases a progressive polynuclear 
leucocytosis is observed. As the tertiary stage comes on the leucocytosis 
usually persists, but the lymphocytosis becomes less distinct and constant 
through the increase in the number of polynuclear cells. ^ 

Justus' Test. 

Justus has found, in studying the blood of patients suffering with florid 
syphilis, that injection or inunction of preparations of mercury cause a reduc- 
tion in the percentage of hemoglobin of from 10 to 20 per cent, for a period of a 
few hours or days. After a certain time, varying with the general condition 
of the patient and the severity of the symptoms, the hemoglobin increases 
again. This test can hardly be considered diagnostic of syphilis, as the mer- 
cury salts all cau3e an anemia which may be directly traceable to their hemo- 
lytic action upon the red cells. 

In the blood of patients suffering with congenital syphilis we always find a 
distinct anemia, associated with a slight leucocytosis, especially of the lympho- 
cytic type. The red cells in this condition show many changes, such as poly- 
chromatophilia and nucleation, while the changes in the white ceUs often 
resemble the picture of a mild grade of leukemia. For a discussion of the 
causative factor, the spirochseta pallida, see the section on Parasitology (p. 63 7) . 

(3) Leprosy. 

The blood in leprosy is quite different from that in either of the two pre- 
vious conditions. The usual rule is a very slight reduction in the number of 
red cells, although cases have been reported with a red count of 1,900,000 
and a blood picture of pernicious anemia. The hemoglobin does not seem to 
be reduced to any extent, the percentage usually being relatively higher than 
the number of red cells, so that a high color index almost invariably obtains. 
The leucocytes are rarely increased in number, being usually subnormal, with 
a relative increase in the number of lymphocytes, their percentage reaching 
as high as 47 per cent., according to Winiarski. 

(4) Carcinoma. 

Although discussed under the heading of Chronic Infections, carcinoma 
has at present no etiological relation to such conditions. This is one of the 
most important causes of anemia, owing to the frequent hemorrhages and the 
mechanical effects of the growth as well as to the unknown toxin, which may 
produce severe constitutional symptoms, even though the growth may have 
become latent. The anemia of malignant disease usually runs parallel to 
the progressive cachexia. The grade of anemia may vary, depending upon 
the location of the tumor, from a very mild chlorotic anemia to one with a 
perfect picture of pernicious anemia. It is natural to suppose that the more 
malignant the disease the greater will the blood changes be, so that we should 

^ See Hazen, Jour. Cutan. Dis., 1913, XXXI, 618 and 739. 



THE BLOOD 617 

expect to find the rapidly growing cancers which form numerous metastases 
usually associated with the more extreme blood picture. That this is not 
always the case is shown by the following statement of Emerson: "Our cases 
with rapidly developing metastases, with large nodules, are those with a slight 
chlorotic anemia; those which simulate pernicious anemia are more often 
those with few objective signs of cancer, an insignificant-looking little 
nodule." It is possible that this paradox may be explained by the fact that 
the development of the cancer is so rapid that the toxin has not had sufficient 
time to cause the blood changes which the more slowly developing growth 
may bring about. The changes in metabolism are much more marked in the 
slowly developing cancers than in the more rapidly growing ones, so that we 
might assume that the same rule applies to the blood changes as expressions of 
the general systemic disturbances. 

When the anemia of cancer develops it is usually more severe than in any 
other chronic disease. The chief changes are at first in the size, shape, weight, 
and degeneration of the red blood-cells; later, as the cachexia develops the red 
cells are often as low as 2,500,000 or even as low as in pernicious anemia, 
1,000,000. The hemoglobin is always reduced in amount, but is rarely as low 
as in chlorosis, the average, according to Cabot, being about 58 per cent. The 
hemoglobin value seems to be lower in cases of visceral cancer than in those of 
peripheral type. In a majority of the cases a moderate leucocytosis obtains 
which is never seen in the benign tumors unless these be complicated by sup- 
puration. This leucocytosis depends largely upon the amount of hemorrhage 
from the tumor and upon the position of the cancer. We find in carcinoma of 
the stomach and uterus, in which hemorrhages are very frequent, quite an 
extensive leucocytosis, while in cancer of the esophagus a leucopenia may ob- 
tain. The larger and faster the tumor grows the greater will be the degree of 
leucocytosis, a condition which is the reverse of that usually found in the case 
of the red cells. The leucocytosis of cancer is usually of the polynuclear type, 
but this may not be over 45 per cent., in which cases the lymphocytes are 
relatively increased. The eosinophiles are rarely as much diminished as 
in other conditions, but they are not always increased. Myelocytes are, per- 
haps, more frequently found in cancer than in the other types of anemia, 
excepting pernicious anemia and leukemia.^ 

The degree of cachexia is often very extensive in cancer, but is not always 
so closely related as one would suppose to the changes in the blood. In those 
cases in which the cachexia is due to a combination of malnutrition with in- 
toxication by the malignant toxin the blood changes are naturally very severe, 
but, as previously stated, the more fulminating types of cancer are not associ- 
ated either with great cachexia or with severe changes in the blood. Cachexia, 
therefore, seems to be more a function of the chronicity of cancer than of its 
malignancy. 

The changes in the blood in the numerous specific diseases of various 
organs show nothing characteristic in themselves. It is to be expected that 
in all chronic diseases of whatever organ, a slight anemia may be present 

^ See von RoznoAvski, Ztschr. f. klin. Med., 1915, LXXXI, 377. 



6l8 DIAGNOSTIC METHODS 

owing to the effects of such disorders upon the general metaboHsm. These 
changes are, however, of the general type of simple anemia and are usually 
rapidly remedied by the application of the ordinary therapeutic agents. It is 
true that disease of the general organs causes changes in the composition of 
the fluid portions of the blood, which may bring about a secondary change in 
the cellular content. 

(e) Effects of Splenectomy. 

The effects of splenectomy are usually the combined results of severe 
hemorrhage, a preexisting anemia, of the loss of functions of this organ, and 
of intravenous infusion^ which has been performed following the operation 
(Ewing) . In comparatively healthy subjects splenectomy has often been per- 
formed without affecting the blood more than does any other abdominal 
operation. Immediately after removal of the spleen an increase in the blood 
pigment and a polycythemia occur (which are probably due to diminished 
hemolysis in the absence of the spleen) providing there is an adequate supply 
of iron in the diet. The most marked changes in the blood are seen in those 
cases in which the organ has been removed for rupture or idiopathic enlarge- 
ment, the loss of blood and the shock of operation giving rise to a consider- 
able degree of secondary anemia. 

The red cells are frequently restored to normal in one to three months, 
due to hyper-activity of the bone-marrow, but in some cases which progress 
less favorably the anemia may be more persistent. The restoration of hemo- 
globin does not take place, as a rule, as rapidly as does that of the red cells. 
Following the operation we usually observe a polynuclear leucocytosis which 
may run, as in one case observed by the writer, as high as 75,000 cells. This 
leucocytosis usually lasts from one to two months, but may persist for several 
months, in which case the polynuclear cells are replaced by lymphocytes. 
Eosinophilia usually develops early and has been observed in some cases two 
or three years after operation. In some cases, especially those suffering from 
extensive hemorrhage, a very profound anemia characterized by great dimi- 
nution in the number of red cells, the presence of polychromatophilic and 
degenerated cells, nucleated red cells, and a high degree of leucocytosis is 
observed. The leucocytes in these cases may take on the picture of an acute 
leukemia, but this condition is transitory as the blood improves more or less 
rapidly. A leucocytosis or permanent lymphocytosis are probably the only 
specific effects of splenectomy. ^ 

1 See Ottenberg and Kaliski, Jour. Am. Med. Assn., 1913, LXI, 2138; Nobel and Steine- 
bach, Ztschr. f, Kinderhkde., 1914, XII, 75. 

2 See Musser, Arch. Int. Med., 191 2, IX, 592; Giffin, Am. Jour. Med. Sc, 1913, CXLV, 
781; Sollenberger, Biochem. Ztschr., 1913, LV, 13; Musser and Krumbhaar, Jour, Exper. 
Med., 1913, XVIII, 487; Pearce and Pepper, Jour. Exper. Med., 1914, XX, 19; Krumbhaar 
and Musser, Ibid., 108; Austin and Pearce, Ibid., 122; King, Arch. Int. Med., 1914, XIV, 
145; Morris, Ibid., 1915, XV, 514; Pearce, Austin and Pepper, Jour. Exper. Med., 1915, 
XXII, 682; Sellards and Minot, Jour. Med. Res., 1916, XXXIV, 469; Mayo, Jour. 
A. M. A., 1916, LXVI, 716; Lee, Minotand Vincent, Ibid., LXVII, 719; Krumbhaar, Ibid., 
723; Miller, Ibid., 727; Friedman and Katz, Ibid., 1295; Schneider, Arch. Int. Med., 1916, 
XVII, 32; Pepper and Austin, Ibid., XVIII, 131; Schneider, Ibid., 1917, XIX, 156; Orr, 
Jour. Lab. and Clin. Med., 191 7, II, 895. 



THE BLOOD 619 

V. Parasitology of the Blood 

(i) Malaria (Paludism; Hemamebiasis). 

Malaria is a disease caused by the entrance of an animal parasite into the 
blood and its development within the red blood-corpuscle. This parasite was 
first studied by Laveran and belongs to the class of sporozoa. It was not 
until the recent work, especially of Grassi, Ross, and Nuttall, that we were 
enlightened as to the source of this invader. It is at present well established 
that the malarial parasite runs its sexual cycle (sporogone) within the body 
of the anopheles mosquito (Anopheles maculipennis).^ The old idea that 
malaria was an air-borne disease, the contagion arising from stagnant pools 
in swampy regions must now be replaced by the modern mosquito theory. It 
is true that the anopheles lays its eggs upon the surface of almost stagnant 
water and that the larvae hatch in these places. The eggs are boat-like in 
shape (each separate, the groups being arranged in ribbons) and float upon the 
surface, while the larvae lie just below the surface and are in a plane parallel 
with it. These facts have led to the adoption of the modern methods of pre- 
vention of malaria by covering the surface of such stagnant pools with oil, 
which prevents access of air to the larvae and, in consequence, causes death. 2 
In a discussion of malaria it must be remembered that there are several 
types of this disease, depending on (i) the kind of parasite causing the in- 
fection and (2) the period at which the various groups of the same parasite 
run their asexual course in the host. We have, therefore, to discuss the three 
types of infecting organism, each of which is a protozoan form and is found in 
the red cells. The cycle of development of the tertian organism is approxi- 
mately 48 hours, so that with a single infection paroxysms will occur on alter- 
nate days. With the quartan organism the cycle of development requires 72 
hours, while with the estivo-autumnal form it is variable, running from 24 to 
72 hours. It is, of course, possible to have infection with more than one form 
of parasite or with more than one series of the same parasite, so that we may 
have daily exacerbations through infection with anyone of these three types of 
parasite. 

In an examination of the blood for the malarial parasite a study of the 
fresh specimen is always desirable when possible, as the pecuUar ameboid 
movements of the parasite as well as the rapid oscillatory movements of its 
granules can, of course, not be seen in the fixed specimen. Moreover, the 
peculiar brassy tone of the red cell and the irregularity in shape and size of 
these cells may best be studied in the fresh specimen. The beginner, however, 

^ Different species of the anophelinae vary in their ability to transmit malaria. While 
more than 100 species are known, many have not been studied. See Walker and Barber, 
Phil. Jour. Sc, B, 1914, IX, 381 ; also, Ludlow, Disease-bearing Mosquitos, Bull. 4, War Dept. 
Office of the Surgeon General, 1914. Recent work has established the fact that anopheles 
punctipennis is an efficient host of the organisms of tertian and estivo-autumnal malaria; 
anopheles crucians of estivo-autumnal type; and anopheles quadrimaculatus of the 
tertian and estivo-autumnal forms. See King, Science, 191 5, XLII, 873; Am. Jour. Trop. 
Med. and Prev. Med., 1916, III, 426; Jour. Exper. Med., 1916, XXIII, 703; Mitzmain, 
Public Health Reports, 1916, XI, 301. 

2 See McCoy, Pub. Health Rep., 1912, XXVII, 1029; also Craig, The Prophylaxis of 
Malaria, Bull. 6, War Dept., Office of Surgeon General, 1914; von Ezdorf, Pub. Health 
Rep., 1913, XXVIII , 2830; Ibid., 1914, XXIX, 503, 613, 871, 1073 and 1289. 



620 DIAGNOSTIC METHODS 

will find that a stained specimen will yield much more definite results, pro- 
viding his staining technic is good than will a study of the fresh specimen, as 
the slight ref ractility both of the cell and of the parasite in the fresh specimen 
makes it difficult in every case to get the proper illumination of the specimen. 
In the hands of an expert the examination of fresh blood is practically all that 
is required for a diagnosis in the average case, and when the parasites are 
moderately numerous the beginner can scarcely make a mistake. It would 
seem, therefore, inadvisable to rest a diagnosis upon an examination of the 
fresh specimen in cases in which no organisms are found, but to control this 
examination by a careful study of a stained specimen in which one may fre- 
quently be surprised at the number of parasites to be seen, although a nega- 
tive result has been observed in the unstained specimen. A word of caution, 
however, is necessary at this point. Frequently one observes in stained speci- 
mens many artefacts due to deposition of staining pigments upon the red cell, 
while in the fresh specimen areas of coagulation necrosis are not infrequently 
seen, so that the untrained observer may assume the presence of malarial 
organisms. For an absolute diagnosis of malaria it is necessary to find intra- 
cellular organisms, and not to be content with a single examination in doubt- 
ful cases. ^ 
Examination of Fresh Blood. 

The technic of making a fresh specimen of suspected blood is the same as 
that previously outlined and consists in touching a perfectly clean cover-slip 
to a drop of blood and allowing the cover-slip to fall upon a clean glass slide. 
The quantity of blood should be rather small so that the red cells may be dis- 
tinctly separated from one another. The examination is best made by the use 
of a J-f 2 immersion lens. 
{a) The Tertian Organism (Hemameba vivax; Plasmodium vivax). 

The youngest form of the tertian parasite as it appears in the red cell 
resembles very closely the spore of the parent rosette. ^ It is a small, compact, 

^Bass has recently succeeded in cultivating the various types of malarial plasmodia. 
See Bass, Jour. Am. Med. Assn., 191 1, LVII, 1534; Bass and Johns, Jour. Exper. Med., 
191 2, XVI, 567; Lavinder, Jour. Am. Med. Assn., 1913, LX, 42. The parasites are cul- 
tivated only in the red cells of human blood and are destroyed iDy the leucocytes as well as 
by the serum. See, also, Sinton, Ann. Trop. Med. and ParasitoL, 1912, VI, 371; and Olpp, 
Miinch. med. Wchnschr., 1912, LIX, 2623. Thomson and Thomson (Proc. Roy. Soc. 
London, 1913, LXXXVII, 77, and Ann. Trop. Med. and ParasitoL, 1913, VII, 153) show 
that cultures of benign tertian parasites differ from those of the malignant tertian forms in 
that there is no tendency to clumping of the former types. This may explain why we do 
not find any but the young forms of the malignant tertian organism in the peripheral blood, 
as the clumping causes the larger older forms to collect in the internal organs. Ziemann, 
Centralbl. f. Bakteriol., i Abt., Orig., 1913, LXVII, 482; Sargent, Beguet and Plantier, 
Compt. rend. Soc. de biol., 1913, LXXV, 324; Bass, Am. Jour. Trop. Dis. and Prev. Med., 
1914, 1, 539. Miller (Jour, Am. Med. Assn., 1914, LXII, 1549) reports the cultivation and 
development of a second generation of parasites without the removal of the leucocytes. 
See, also, Clarke, Lancet, 191 7, I, 530. 

2 Stephens (Proc. Roy. Soc. London (B), 1914, LXXXVII, 375, and Ann. Trop. Med. 
and ParasitoL, 1914, VIII, 119) reports the discovery of a liew malarial parasite, which he 
names Plasmodium tenue. Craig (Jour. ParasitoL, 1914, I, 85) believes this to be nothing 
more than a rather atypical form of Plasmodium vivax in the unpigmented stage of 
development. Lawson (Jour. Exper. Med., 1916, XXIV, 291) regards the Plasmodium 
tenue as not a new type but a parasite attached to the external surface of red cells and dis- 
torted by technic. 

Emin (Bull. Soc. Path. Exot., 1914, VII, 385) describes a further variety of the tertian 
organism to which he gives the name Plasmodium vivax variety minuta Craig does not 
believe the evidence sufficient to give this parasite specific rank. 



PLATE XXVIII. 



10 11 



^^ 



X 



12 




•V-??.!* 

» 



14 



p 



The Tertian Parasite. 



1. Normal erythrocyte. 

2, 3, 4, 5. Intracellular hyaline forms. 

6, 7. Young pigmented intracellular forms. In 6 two distinct parasites inhabit the ery- 
throcyte, the larger one being actively ameboid, as evidenced by the long tentacular 
process trailing from the main body of the organism. This ameboid tendency is 
still better illustrated in 7, by the ribbon-like design formed by the parasite. Note 
the delicacy of the pigment granules, and their tendency toward peripheral arrange- 
ment in 6, 7, and 8. 

8. Later developmental stage of 7. In 7, 8, and 9 enlargement and pallor of the infected 

erythrocyte become conspicuous. 

9. Mature intracelhilar pigmented parasite. 

10,11, 12. Segmenting forms. In 10 is shown the early stage of sporulation — the develop- 
ment of radial striations and peripheral indentations coincidentally with the swarm- 
ing of the pigment toward the center of the parasite. The completion of this process 
is illustrated by 11 and 12. 

13. Large swollen extracellular form. Note the coarse fused blocks of pigment. (Com- 

pare size with that of normal erythrocyte, i.) 

14. Flagellate form. 

15. Shrunken and fragmenting extracellular forms. 

16. Vacuolation of an extracellular form. 

Note. — The original water-color drawings were made from fresh blood specimens, ?. 
Leitz i*2-inch oil-immersion objective and 4 ocular, with a Zeiss camera-lucida, being used. 



(E. F. FABER.y^t.) 

(Prom Da Costa's "Clinical Hematology ") 



THE BLOOD 62 1 

colorless, non-pigmented disk (hyaline form) about 2 microns in diameter and 
shows an undulating outer rim of basophiUc protoplasm which encloses a single 
large nuclear body which does not stain with methylene blue but shows a dis- 
tinct chromatin stain with any of the modifications of the Romanowsky 
stain. This nuclear body is usually surrounded by a clear space which does 
not take the stain and which has been termed by Gautier ''the milky zone.'' 
The parasite has a very rapid ameboid movement and shows a great number 
of changes in shape and position. It sometimes assumes a typical ring-like 
form which is usually a little thicker at one point, from which the name ''signet 
ring'' has been given. Occasionally several of these rings may be seen within 
a single blood-cell. After about 12 hours the corpuscle increases slightly in 
size, becomes somewhat paler, but still has the sharp, smooth, round outline 
of the normal cell. At this stage the ameboid powers of the organism are very 
marked, so that many pseudopodia may be seen, connected to the .larger part 
of the organism by very thread-like pale and rather indistinct bands of union. 
This gives the appearance of disconnected globules of protoplasm, which is 
very slightly refractile. At this period (12 hours) pigment (hematin) appears 
in the parasite in the form of very fine light brown granules which have a very 
rapid dancing motion and are clustered especially at the ends of the pseudo- 
podia. The organism continues to increase in size and, at the same time, the 
host becomes somewhat larger, paler, but still round in outline. At the end 
of 24 hours the organism fills about one-third of the cell, is still ameboid and 
shows increased pigment, which is somewhat darker in color and is less ac- 
tively motile, being distributed throughout the substance of the parasite. In 
this form one may occasionally see the nucleus as a globular body at the end 
of a pseudopod. In the last half of the cycle of development of the tertian 
organism the growth is much more rapid than in the first half, the parasite be- 
ing fully developed within 40 hours. The cell at this time is about one and a 
half times its normal size and is so little refractile that its outline can scarcely 
be seen. The organism is from 8 to 10 microns in diameter, is round, and is 
even less refractile than is the corpuscle. The pigment is much more abun- 
dant at this time and is still evenly distributed throughout the organism. The 
next stage in the development of the organism is known as the presegmenter 
stage. The cell becomes practically invisible, the pigment collects in one or 
more irregular clumps throughout the organism, the granules moving in irregu- 
lar lines. At this time the periphery of the organism shows slight crenation 
and refractive dots appear irregularly in the periphery of the organism. The 
line of demarcation between the presegmenter and the segmenter is very 
slight. The corpuscle is now practically eliminated and the organism be- 
comes more dense and highly refractile. The refractive dots which were 
visible in the presegmenter stage are now seen to be in the center of lines of 
separation which pass from the irregular crenated border down toward the 
center of the organism, thus marking off future segments, which are as a rule 
from 15 to 20 in number. As development proceeds the segments become 
more sharply defined until the clumps form into discrete circular masses with 
a distinctly refractile spot in the center. The pigment in these segment forms 



622 DIAGNOSTIC METHODS 

seems to be left in masses between the segments without any definite arrange- 
ment. Each segment now spHts off from the mother segmenter cell and 
becomes free in the blood in the form of the original hyaline type which be- 
comes attached to a red cell and soon enters it to pass through the vari- 
ous stages discussed. It is to be remembered that the hyaline forms do not 
modify their host, either in shape, color, or size, such changes being observed 
only at the time when the pigment first becomes evident. 

The preceding is a concise description of the cycle of development within 
the cell {asexual generation or schizogone), but we occasionally find tertian 
forms other than hyaline which are extracellular. These seem to be of two 
types, the degeneration forms and the gametocytes, cells capable of sexual 
development. The degeneration forms or, as they are sometimes called, ex- 
truded intracellulars, are sometimes the only ones seen in the specimen. 
These are parasites which have passed from the cell and have died, the organ- 
ism sometimes appearing as if it had passed out through a very fine hole. If 
it be entirely extruded, the hemoglobin leaves the cell with it and only a 
shadow of the red corpuscle remains behind. However, this does not always 
occur, so that we meet with typical dumb-bell-shaped organisms in the plasma. 
If the blood be observed while this process of extrusion is going on, the 
pigment will still be extremely active but it gradually becomes quiet as the 
organism dies. The organism may break up into fragments forming several 
pigmented spherical masses or it may become deformed and vacuolated, con- 
stituting the so-called "sporulating" forms. The gametocytes are found at 
all times in the blood after the infection has been established for a few days. 
These are not in reality extracellular forms as one sees them in the stained 
specimens surrounded by the shell of the corpuscle. These gametocytes are 
of two forms, the macro gamete or female cell and the micro gamete, the male cell, 
which is one flagellum of the micro gametocyte (parent male cell) . The macro- 
gametes are large organisms, pale, indistinct, and three or four times as large 
as the red cell. Some of them show no trace of the corpuscle; their pigment 
is abundant and exhibits very active movements. Their nucleus is about 
three and one-half microns in diameter and is sometimes seen in the fresh 
specimen; either its outUne is distinct or its size and shape may be recognized, 
as it is the only portion of the parasite which is not invaded by the pigment 
granules. The function of these macrogametes seems to be to continue the 
life of the organism within the mosquito after the organism has become fertil- 
ized by the male element or microgamete. The microgametocyte is smaller 
than the macrogamete, being eight to ten microns in diameter. Its pigment 
is very active, but soon forms a circle around the center and becomes sta- 
tionary. Occasionally this pigment may become even more active than be- 
fore, the margin of the cell may undulate and several flagella protrude. 
These flagella are the microgametes and are two or three times the length of a 
red blood-cell and often contain pigment granules which enable them to be 
followed when they break loose from the parent cell. After these flagella 
separate, the parent cell is seen as a small cell with central motionless pigment. 
This process of flagellation is not seen in the fresh specimen, but occurs 1 5 or 



PLATE XXIX. 



^ 



^1 



o * . » ' 



10 11 






The Quartan Parasite. 



1. Normal erythrocyte, 

2. httracellular hyaline form. 

3. Youuff pigmented intracellular form. Note the coarseness, dark color, and scantiness 

of the pigment granules. 

4. 5, 6, 7. Later developmental stages of j. Note the peripheral distribution of the pigment 

in all the parasites from 3 to 8. (Compare size and color of the erythrocytes in 5, 6, 
and I with 7, 8, and 9, Plate VI.) 

8. Mature intracellular form. Note that the stroma of the erythrocyte is no longer 

demonstrable. 

9, 10, II. Segmenting forms. In 9 are shown the characteristic radiating lines of pigment. 

(Compare with 10, 11, and 12, Plate VI, and with 10, 11, and 12, Plate VIII.) 

12. Large swollen extracellular form. (Compare with 13, Plate VI.) 

13. Flai^ellate form. (Compare with 14, Plate VI.) 

14. V'acuulation of an extracellular form.. 

(E. F, Faber,/<?c.) 
(From Da Costa's "Clinical Hematology.") 



THE BLOOD 623 

20 minutes after the blood has been drawn, which would point to the fact that 
such a process does not occur within the body. Normally, this change takes 
place only in the stomach of the mosquito. 

(b) The Quartan Organism (Hemamebamalarise ; Plasmodium malariae)- 

This organism is much more rare than is the tertain. Its cycle of develop- 
ment requires 72 hours so that the normal paroxysm occurs every fourth day. 
If two groups are causing the infection there will be two days with paroxysms, 
one free day, and then two days of paroxysm following. If more than two 
groups are introduced we may have daily chills and fever, but only when the 
groups are large enough in number to cause a paroxysm. According to Ross, 
250,000,000 organisms are necessary before a chill follows. The small hya- 
line forms of the quartan parasite are not distinguishable in the early stages 
from those of the tertian type, but are easily recognized at the time pigment 
appears, as the granules of the former are coarser, darker in color, and not so 
actively motile. As the parasite grows in size the corpuscle becomes smaller 
and stunted with an irregular crenated margin. The protoplasm of the 
organism is more refractile than that of the tertian organism and hence the 
outlines of the pseudopodia are more easily seen, although the parasite is 
less actively motile. In 24 hours the red cell is quite small, crenated, and 
distinctly brassy in color. The organism is round or oval, quite distinct, 
slightly ameboid, and its pigment blackish-brown in color and gathered at the 
periphery, especially on one side, thus differing from the tertian organism 
in which the pigment is scattered throughout the organism. The pigment 
granules have practically no motion at this stage of the development. As 
development proceeds the parasite fills 'from one- third to one-haK of the cell, 
becomes rounder, and loses its ameboid power. The protoplasm is very 
distinct and highly refractile. During the third day only a rim of the cell 
is left and this usually takes on a dark, brassy tone. The organism is at 
this time full grown and is about seven microns in diameter. The pigment 
now passes from the periphery of the organism toward the center in definite 
radial lines, giving a wheel-like formation, with the pigment granules forming 
the spokes. Later the pigment collects in the center and we have the forma- 
tion of the presegmenter form. Following this the organism becomes opaque, 
refractive dots appear in a single regular circle about the periphery, and 
crenations of the border appear with these dots as a center. Lines of divi- 
sion start from these crenations and run to the center, forming from six to 
twelve segments, like the petals of a flower, giving rise to the name "daisy," 
''marguerite," or "rosette" form. These quartan segments are much more 
perfect than are the tertian forms, and later separate to form the hyaline types 
which take up the development as outlined above. This whole cycle of the 
development of the quartan organism takes place in the peripheral blood 
as it does in the tertian organism, but the number of segmenter forms is 
much more numerous in the quartan type than it is in the tertian form. 
This is probably due to the fact that a large number of the tertian forms 
accumulate in the internal organs. 



624 DIAGNOSTIC METHODS 

The gamete forms are not as frequently seen as are those of the tertian 
organism. They are similar in appearance, but somewhat smaller than those 
of the tertian organism and give rise to flagellation in the same manner. The 
extracellular forms are occasionally found, but not so frequently as those of 
the tertian form. 

The distinguishing marks between these two types of organisms may 
be summarized as follows : The cycle of development of the tertian organism 
is 48 hours, while that of the quartan is 72. The quartan organism is smaller, 
more refractile, less ameboid, and its pigment is coarser, darker, less motile, 
and more peripheral in position. The corpuscle infected by the quartan 
organism is smaller, shrunken, crenated, and more brassy. The presegmenter 
and segmenter stage are much more distinctive in the quartan than in the 
tertian type and more of the segment forms of the former are found in the 
peripheral blood, although the number of segments of the quartan type are less 
than those of the tertian parasite (Emerson). 

(c) The Estivo-autumnal Parasite (Plasmodium precox; Plasmodium 
falciparum). 

This is the most dangerous type of malarial infection. The duration 
of the cycle of development varies from 24 to 72 hours. In infection with 
this organism the members of the same group do not always develop in the 
same unity, so that we may find at times an intermittent fever, but one which 
becomes more and more continuous. The hyaline forms are similar to those 
of the tertian and the quartan types, but are slightly smaller and assume the 
"signet-ring" form much more commonly and maintain it longer. This 
early form of the estivo-autumnal parasite is distin.guishable from the tertian 
by the shrinkage of the red blood-cell and from the quartan parasite by the 
smaller dimensions. In some cases the rings do not show the thickening of 
one segment, but remain of a uniform fine caliber throughout. These rings 
which do not show the distinct ''signet-ring" type nearly always present two 
nuclear bodies lying at opposite poles or close together. Occasionally these 
rings appear as if unfolded and stretched across the cell like a thread, the 
nuclei appearing at irregular intervals. These rings may at times lose their 
refractility and become ameboid. As the parasite develops, a slight amount 
of pigment appears, usually seen as one or two granules which are motionless, 
as a rule, and are located at the periphery of the parasite or at the inner 
edge of the biconcavity. The cell is very commonly much shrunken, crenated, 
and brassy, even in the early stages. Some cells, which do not contain parasites, 
show the same injurious effects of the organism. The parasite at this time 
occupies about one-fifth of the cell. The infected cells now usually disappear 
from the circulation and continue their development in the lymph-glands, 
especially in the spleen. In some cases, however, the parasite does continue 
its development in the peripheral blood, but this is rare. In such blood or in 
that obtained from the spleen, the pigment appears much increased and seems 
to be rather coarse and dark in color, thus resembling very closely the quar- 
tan organism at this stage. It seems to be a general rule that the more malig- 



PLATE XXX. 



4 5 



t * ^ ^ «.^ 



-s^T 



10 11 

8 9 12 13 14 



5 



4S 
15 16 17 18 



? A ^* -^^ 



19 20 21 22 



^^ # 



€. 



24 

23 &^^ 26 

?^ 25 



The Estivo-Autumnal Parasite. 



1. Normal erythrocyte. 

2, 3. Voung hyaline ring-forms. 

4, 5, 6. Intracellular hyaline forms. In 4 the parasite appears as an irregularly shaped disc 
with a thinned-out central area. In 5 and 6 its ameboid properties are obvious. 

7. Young pigmented intracellular form. Note the extreme delicacy and small number of 

tne pigment granules. (Compare with 6, Plate VI, and with 3, Plate VII.) 

8, 9. Later developmental stages of 7. 
10, II, 12. Segmenting forms. 

13, 14. Crescentic forms at early stages of their development. 

15, 16, 17, 18, 19. Crescentic forms. In 15 and 19 a distinct " bib " of the erythrocyte is visible. 
Vacuolation of a crescent is shown in 18, and polar arrangement of the pigment in 17. 

20. Oval form. 

21, 22. Spherical forms. 

23. Flagellate form. 

24. Vacuolation and deformity of a spherical form. 

25. Vacuolated leucocyte apparently enclosing a dwarfed and shrunken crescent. 

26. Remains of a shrunken spherical form. 



(E. F, Faber,/^c.) 
(From Da Costa's "Clinical Hematology.") 



THE BLOOD 625 

nant the type of estivo-autumnal malaria, the fewer older forms are seen in 
the peripheral blood, although numerous young parasites are present. In some 
cases the hemoglobin becomes concentrated around the parasite, leaving an 
almost colorless ring at the periphery of the cell. The cycle of development 
in the internal organs seems to take place within the macrophages, which are 
best studied in the fresh specimen. The parasite develops to about 5 microns 
in size, which is about half the size of the cell, and when full grown has its 
pigment all in the center, never diffusely scattered as in the tertian organism 
or peripherally located as in the quartan type. This form is rarely seen in 
the peripheral circulation. The segmenters vary in size from 2)^ to 5 
microns in diameter, the process of segmentation being similar to that of the 
other organisms giving rise to the formation of 15 or 16 very small segments. 
Certain characteristic forms of this type of malaria appear in the pe- 
ripheral blood from about the seventh day of infection and in the internal 
organs as early as the fifth day. These forms are known as the crescents 
and the avoids. The crescents^ are slightly longer than the red blood-cells 
and show a distinctly crescentic shape with rounded ends, although irregular 
forms are at times observed. They are very refractile and usually show a 
fringe of the degenerated red blood-cell, which is more abundant in the con- 
cavity of the crescent and forms the so-called "bib." The pigment is large 
in amount and is massed at the center of the crescent, occasionally in the form 
of a sheaf or ring. The granules are usually coarse and rod-shaped. These 
crescents very frequently change their shapes, becoming oval, dumbbell- 
shaped, or circular and then may resume their original crescentic form. In 
the circular types no trace of the corpuscles is seen and the protoplasm is not 
as distinctly refractive as is that of the crescent. In this form of malaria we 
also find pigmented leucocytes, both the polynuclear neutrophiles and the 
large mononuclears assuming this function. In these phagocytic cells one 
may see masses of pigment or even parasites, especially the segmenting 
and flagellating forms. These pigmented cells are also seen in the other 
form of malaria, but only just after the chill, while in the estivo-autumnal 
form they may occur at any time during the infection. 

Examination of Stained Specimens. 

The technic of making preparations of malarial blood for staining is 
practically the same as that outlined previously. Precaution must be taken 
tdmake thin smears so that the parasite may be brought out more clearly. 
The stains to be used will depend largely on the experience of the worker, but 
the writer would recommend the thionin and Nocht stains above the others, 
although the Wright and Giemsa stains will frequently give beautiful pictures. 
Stains which have been kept for some time are not always reliable, so that it is 
well to have fresh specimens of the stain on hand for use. If the blood has 
been kept for some time before staining a diffuse plasma staining with 
methylene blue will be observed. 

_ ^ See Thomson (Ann. Trop. Med. and ParasitoL, 1914, VIII, 85) for a discussion of the 
origin and development of these crescents. 

40 



626 DIAGNOSTIC METHODS 

The Tertain Parasite. 

The young hyaline form consists of a mass of blue protoplasm usually 
grouped in ring-form with a mass of reddish-violet stained chromatin, usually 
situated at the thinner portion of the ring and extending for a large part within 
the clear achromatic or vesicular part of the parasite. These hyalines are 
2 to 3 microns in diameter. There is some discussion as to what portion of 
the parasite the name nucleus should be applied. Some give this term only 
to the chromatin staining part, while others include both the chromatin and 
achromatic portion. However this may be, it is necessary for the recognition 
of the tertian organism that the blue protoplas m and the red chromatin be 
both observed. There are frequently artefacts in the blood which resemble 
very closely these hyaline forms so that the worker must be constantly on his 
guard. Such artefacts are the Maragliano degenerations so commonly seen 
in red blood-cells and also the cases in which blood-platelets lie upon a 
red blood-ceU. Any structure which lies upon a red blood-corpuscle ap- 
pears surrounded by a colorless zone, while the true malarial ring is in direct 
contact with the hemoglobin of the cell. Moreover, such artefacts will not 
show the chromatin staining portion which is so characteristic of the hyaline 
ring. In the specimens examined at the end of 24 hours, one will observe 
that the achromatic area has become somewhat larger, while the chromatin 
portion seems to be grouped in more irregular masses, some cells appearing 
to have several nuclei. In the full-grown parasite the chromatin breaks 
up into a cluster of fine granules which are scattered diffusely through the 
cell in the form of strands and masses. These chromatin clumps separate 
into from 15 to 20 dense round masses, around which the protoplasm collects 
with them as a center. The protoplasm at this stage is distinctly achromatic 
and is always so in the segmenting cell. The distinct, achromatic, milky 
zone surrounds each segmentary chromatin clump, while the general proto- 
plasm shows a diffuse faintly basic staining. The pigment which must not be 
confused with the chromatin is pushed toward the periphery and, after 
segmentation is complete, collects in masses near the center. It is to be re- 
called that at the time the pigment collects in the. center in the fresh specimen 
there is no distinct evidence of segmentation, although this segmentation 
shows quite distinctly in the stained specimen. 

The sexual development of the parasite is easily followed in some cases 
in the stained specimen. According to Stephens and Christophers, the young 
gamete is characterized by the position of the chromatin as it lies in the 
center of the vacuole instead of at the edge. During the development the 
gamete is occasionally filled with basophile particles which are known as 
Plehn's karyochromatophilic granules or Schligner's granules. The full- 
grown macrogamete contains an abundance of protoplasm which stains a 
deep blue and a small amount of chromatin in a compact mass, which is 
peripherally placed and surrounded by a thin vacuole-like area. The pig- 
ment of these female cells is uniformly distributed throughout the cell and the 
inclosing red blood-cell can be seen only with difficulty. The chromatin is 
much more voluminous in the microgametocytes, but is looser and centrally 



PLATE XXXI. 




Tertian Malarial Parasite. (Wright's Stain.) 



PLATE XXXII. 




ESTIVO-AUTUMNAL PARASITE. (WrIGHT'S StAIN.) 



THE BLOOD 627 

placed in a large achromatic zone arranged in the form of a band which 
stretches clear across the cell. The protoplasm is in the form of a ring 
around the nucleus and stains more of a grayish-green color than does the 
bluish protoplasm of the macrogamete. 

The Quartan Parasite. 

The structure of the quartan parasite resembles very closely that of the 
tertian form, but in the hyaline type the chromatin mass is less distinct and is 
in the form of an irregular clump of granules in the older forms, while in the 
younger a cluster of fine granules without any distinct achromatic zone is seen. 
As development proceeds the parasite generally takes a form extending across 
the cell and usually occupies the larger portion of the red cell which has be- 
come shrunken and irregular in shape. The segment forms are much more 
distinct in the quartan type than in the tertian and show much more regular 
and geometric lines of cleavage with the chromatin exactly in the center of 
the crenated surface. The pigment granules are coarser and much more dis- 
distinct than in the tertian form and are more peripherally located. 

The Estivo-autumnal Parasite. 

The hyaline forms in this type show the chromatin in two or more masses 
or filaments. The protoplasm is scantier than in the other forms and remains 
so throughout the cycle of development of this parasite. A very character- 
istic appearance of the hyaline rings of this type seems to be the thickening 
of the protoplasmic layer opposite the chromatin mass. The gamete forms 
are distinctly spherical, being of the same thickness all the way round. Their 
nucleus forms a portion of the ring, but does not project as in the schizonts 
(the asexual parasites). The red blood-cells in these sexual types usually 
show no coarse granular stippling . The crescent forms, which are character- 
istic of this type of malaria, show the chromatin in a loose network which 
occupies the larger portion of the cell, has little blue staining protoplasm, 
and has its pigment scattered throughout its body. This is the male form 
and is somewhat kidney-shaped and is shorter and broader than the female 
type. The female crescent is longer and narrower, its chromatin more or 
less compact and centrally located, its pigment in a ring around the nucleus 
or in a clump at the center, while its protoplasm is more or less extensive and 
takes a distinct bluish tinge . We also find two types of the circular form. 
The microgametocyte is smaller than the red cell, distinctly spherical in 
shape, with its chromatin in the center in a large irregular mass, or in several 
dense masses near the periphery. These masses containing chromatin material 
are later extruded and form the flagella or microgametes. The macrogamete 
is two or three times the size of the microgametocyte, is often of a triangular 
shape, and has abundant blue-staining protoplasm. The chromatin is in a 
single mass at the periphery and is surrounded by a circle of pigment. 

The examination of the stained specimens does not give as great an oppor- 
tunity for study of the developmental cycle of these parasites as does the 
examination of a fresh specimen. The conditions found in the fresh blood 
resemble more nearly those found in the stomach of the mosquito than in 



628 



DIAGNOSTIC METHODS 




c b 

Fig. 143. — Cycles of the Malarial Parasite. {Deguy et Giiillaumin.) 
I, Beginning of the development of the parasite as the ameboid intra-cellular body. 
/, //, ///, and IV represent the endoglobular cycle or schizogony. /, //, //', //", ///", 
and ///' represent the extra-cellular cycle or sporogony. In //', there are two free gametes 
{g), one microgametocyte {m), one microgamete {m'), and the union of a macrogamete 
and a microgamete {m"). Ill', free gametes; ///", fertilized macrogamete, taken up by 
the mosquito, A. lnB,C, D and E it becomes encysted in the gastric musculature forming 
the zygotes (z and 2'). In E, the sporozoits are formed and in F they are thrown out by 
the saliva of the mosquito. In G, these are free to enter the cell forming the ameboid 
bodies. 



THE BLOOD 629 

the circulating blood, so that many pictures seen in the fresh specimen are 
practically never found in the stained slide. ^ It is further to be said that we 
do not always find malarial organisms either in the fresh or stained specimen, 
although the patient may be at the time suffering from malaria. It may be 
stated, as a rule, that in all well-marked initial attacks of malarial fever the 
parasite may be found in the blood if it be examined within 18 hours of the 
chiU. The energetic use of quinin has so much influence upon the ameboid 
types of the parasites, that the blood may fail to show any of these organisms, 
although the patient may die from the effect of the infection. It is safe, how- 
ever, to state that there is practically no case of malaria in which parasites may 
not be found in the blood, if frequent and repeated examinations are made. 

Development of the Organism within the Mosquito (Sporogony). 

The cycle of development of the malarial organism has been more closely 
followed in the mosquito in the case of the estivo-autumnal parasite. For 
any development to occur within the body of the mosquito it is necessary 
that the macrogamete become fertilized, so that the course in the mosquito 
is one of sexual development. The microgametocytes throw out their flagella 
(microgametes) and the macrogamete ripens in the stomach of the mosquito 
by casting off karyosomes (polar bodies consisting of chromatin), and in so 
doing causes the formation of a slight mound at one portion of the organism 
through which the free flagellum enters. This process occurs in from one to 
one and a half hours after the mosquito has bitten a patient infected with 
malaria. The nuclear material of the macrogamete and microgamete then 
unite. The cell then forms a distinct motile spindle shape called the ver- 
miculus or ookinet. The size of this fertilized macrogamete is from 20 microns 
up and may be found in about 48 hours after the blood has been ingested. 
This motile form is found only in the stomach of the mosquito. The ver- 
miculus then bores its way through the epithelial cells of the intestinal wall 
and becomes encysted between the intestinal epithelium and the elastic layer, 
which forms the membrane of the spore cyst (zygote, oocyst, sporohlast). This 
zygote increases rapidly in size and the nucleus divides rapidly. In its growth 
it bulges outward from the intestinal wall forming pendulous tumors into the 
body cavity which growth may vary from four and a haK to ninety microns 
in diameter. This stage is associated with the appearance of much pigment 
and is called the medium zygote or medium sporoblast stage. The protoplasm 
gathers around the divided nuclei, forming daughter cysts which are con- 
nected by bridges of protoplasm forming the stage known as large zygote, large 
sporohlast, or large oocyst. In each of these divisions the nucleus divides 
many times, the daughter nuclei remaining on the surface of each daughter 
cyst. The protoplasm now collects around each daughter nucleus, the first 
forming spherical cells which then elongate into threads lying parallel over 
the remains of the sporoblasts. These threads are called sporozoits and have 
an elongated nucleus. The final length of these sporozoits is about 14 
microns and their width about i. Their protoplasm is thick, homogeneous, 

^ See Clark (Jour. Exper. Med., 1915, XXII, 427) for a discussion of the value of the 
study of placental blood films in the diagnosis of malaria. 



630 DIAGNOSTIC METHODS 

and very refractive. They are sometimes present to the number of 10,000 
in some zygotes, but more frequently are not so numerous. As the oocyst 
becomes larger it bursts into the body cavity, the sporozoits of each cyst 
ripening at about the same time. These sporozoits wander at first free, but 
soon collect in the salivary gland of the mosquito. They are motile and move 
with a bending and gliding motion. When they are inoculated into the blood 
of man by the bite of the female mosquito they attach themselves to the red 
blood-corpuscle and finally penetrate it to form the initial hyaline type 
of the organism. The period of incubation after the bite of the mosquito 
is usually between the eighth and twelfth day, when the first chill will 
appear, although the exact time of appearance of the initial symptoms will 
depend upon the number of sporozoits introduced into the circulation (Emer- 
son) . The anopheles is the only type of mosquito which is at present known 
to be the host of the malarial organism and to give rise to the development of 
the gameto-schizonts (the sexual cells), which the bite of the female animal 
introduces into the blood cells which are known in their future development 
as schizogones. The sexual cycle within the mosquito is known as the sporo- 
gone, which Rowley-Lawson^ has recently shown may occur in the circulating 
blood of the human host. 

General Changes in the Blood in Malaria. 

There are few conditions which lead so rapidly to such an extreme reduc- 
tion in the red cells as does acute malaria. 2 An acute attack may reduce the 
red cells to as low as 500,000 cells as reported by Kelsch. Frequently a re- 
duction of 1,000,000 is observed during the first day, with a progressive reduc- 
tion as the time goes on. In the afebrile period of the disease a continuous 
fall is observed, but this is much less rapid. The regeneration of the cells is 
very active so that an increase in the number of cells has been observed 
directly after an attack in some cases. In cases of chronic malaria the red 
cells are commonly reduced to as low as 583,000 (Kelsch), while when attacks 
occur only at intervals and are promptly stopped by quinin no reduction in 
the red cells may follow (Marchiafava). In cases of moderate severity the 
usual changes of secondary anemia are present in the red cells. Polychro- 
matophilia and granular degeneration of the reds progresses steadily, while 
the hemoglobin content of the cells may be markedly reduced. Frequently 
cases are seen in which the anemia takes on the absolute pernicious type, so 
that the parasites seem to have been massed in the bone-marrow. As Ewing 
states, 'Hhere can be no doubt that the tendency of the estivo-autumnal 
parasite to be massed in the bone-marrow, in both ameboid and crescentic 
phases, and the excessive demand on red-cell production arising in the disease 

1 Jour. Exper. Med., 1911, XIII, 263; Ibid., 1913, XVII, 324. See, also. King, Jour.' 
Exper. Med., 191 7, XXV, 495. 

2 Rowley-Lawson (Arch. Int. Med., 191 2, IX, 420) believes that this severe anemia is 
due to the migration of the plasmodia from corpuscle to corpuscle. See James, Jour. Infect. 
Dis., 1913, XII, 277; Brown, Arch. Int. Med., 1913, XII, 315; Jour. Exper. Med., 1913, 
XVIII, 96; Rowley-Lawson (Ibid., 1914, XIX, 450 and 453; Ibid., 1915, XXI, 584) dis- 
cusses the extra-cellular relation of the parasite to the red corpuscle. See, also, Lawson, 
Jour. Exper. Med., 1918, XXVII, 739 and 749; Ibid., 1919, XXIX, 361; Cardier, C. R., 
soc. biol. Paris, 1919, LXXXII, 355. 



THE BLOOD 631 

render pernicious malaria an extremely favorable condition for this disturb- 
ance of the structure of the marrow and the development of specific megalo- 
blastic changes." Besides the changes which can be directly referred to 
anemia or toxemia, changes in the size of the cell are quite constant, the ter- 
tian parasite causing from the start swelling of the cell and progressive loss of 
hemoglobin, while the quartan and estivo-autumnal forms cause the red cell 
to shrink and take on a peculiar brassy tone. 

The leucocytes do not show very characteristic changes. In the acute 
malarial attacks of average severity the absence of leucocytosis is of consider- 
able corroborative value, although a slight leucocytosis amounting to about 
10,000 with an increase in the percentage of polynuclear cells has been ob- 
served by Billings and others. Except during the three or four hours im- 
mediately following a chill malarial blood usually shows a diminished number 
of' leucocytes with a distinct relative lymphocytosis, which finding is that 
seen in typhoid fever. ^ In the more severe estivo-autumnal attacks a definite 
leucocytosis has been distinctly observed, especially in the hemoglobinuric or 
black-water type of malarial infection. The extent of the leucocytosis varies 
between 10,000 and 35,000, although many attacks fail to cause any distinct 
increase. During the afebrile periods the eosinophile cells are usually in- 
creased and may be observed throughout the course of the attack. Neutro- 
phil myelocytes are occasionally present and rarely eosinophile myelocytes. 
Pigmented leucocytes are seen in the majority of cases, especially in the severe 
and fatal cases, the pigmented leucocytes being more closely related to the 
severity of the paroxysms than to the extent of the deposits in the various 
viscera. These pigmented or phagocytic cells include mononuclear and poly- 
nuclear leucocytes and a few endothelial cells. The large and small mono- 
nuclears usually contain pigment or rosettes, while many of the polynuclear 
leucocytes also contain the parasites. These phagocytes may contain, be- 
sides parasites and malarial pigment, hematoidin, hemosiderin, red blood- 
cells, leucocytes, and occasionally an unknown crystalline pigment. 

(2) Relapsing Fever (Famine Fever). 

The cause of this fever is the spirillum of Ohermeier {spironema recurrentis) 
and is not a member of the class of bacteria but belongs to the class of spiro- 
chete. This organism is between 16 and 40 microns in length and about i 
micron in width, but is subject to considerable variation in size. It is thin,^ 
sharply curved, and appears to be structureless. It takes a deep chro- 
matin stain and also stains with methylene blue in from two to five minutes. 
It is seen in the blood only during the febrile period of the disease and 
at that time is actively motile with a rapid wavy motion, much resembling 
the movements of a coiled spring in its stretching and collapsing. It moves 
rather slowly among the corpuscles, but does not disturb them to any extent.^ 
Cases have been reported in which these spirochete are present in the blood 

^ See Zweig and Matko, Wien. klin. Wchnschr., 1916, XXIX, 1328. 

2 Noguchi (Jour. Exper. Med., 191 2, XVI, 199) has succeeded in cultivating this organ- 
ism and shows that this pathogenicity is not thus diminished. See Plotz, Ibid., 191 7, X— 
Xyi, 37- 



632 DIAGNOSTIC METHODS 

24 hours before the chill, but they are usually to be found in larger numbers 
at the time of the rise in temperature, increasing rapidly from day to day. 
The fever, as a rule, continues about six days, at the end of which time these 
parasites leave the blood. Strangely enough these organisms have been 
found in varying numbers in different parts of the circulation, while there 
does not seem to be any strict parallelism between their number and the 
height of the fever. Loewenthal has applied the agglutination test to the 
blood of suspected cases and finds the reaction positive in 85 per cent, of the 
cases in the periods in which the parasites are absent. Nothing of a charac- 
teristic nature is observed in the general blood picture. 




Fig. 144. — Spirillum of Obermeier. {Pit field.) 

Cases of relapsing fever are practically never found in the United States, 
unless imported through the medium of emigrants from Russia and especially 
India. The cases reported by Wellman show that relapsing fever, as found 
in West Africa, may arise from the bite of a tick transmitting the spirillum 
of Obermeier.^ 

(3) Sleeping Sickness. 

This very interesting condition which is so prevalent in Central and West 
Africa seems to be due to an actively motile fusiform flagellate known as the 
trypanosoma Gambiense, which can be found in the blood free in the plasma 
(never intracorpuscularly), moving with a screw-like motion among the red 
cells which it does not seem to disturb. This parasite doubtless has a sexual 
development, its host being the common fly, Glossina palpalis, while closely 
related trypanosomata are transmitted by the bites of various flies, especially 
one of the seven varieties of the tsetse fly. This organism is from two to 
three times as long as a red blood-corpuscle (18 to 25 microns) and 2 to 2}/^ 
microns wide, having a flagellum anteriorly and an undulating membrane ex- 

1 Nicolle, Blaizot and Conseil (Ann. de I'lnst. Pasteur, 1913, XXVII, 204) show that the 
bite of the louse does not transmit this disease as is commonly supposed. If the louse be 
crushed on the skin and there are abrasions on the skin from scratching, the parasites gain 
access to the system. 



THE BLOOD 633 

tending its entire length.^ In the fresh blood specimen these parasites should 
be looked for with only a medium magnification. These parasites vary much 
in number sometimes being absent for a long period and then suddenly re- 
appearing in large numbers. Symptoms of the disease seem to bear little 
relation to the number of parasites in the peripheral blood, so that in some 
cases it may be necessary to examine the fluid in the edematous areas or even 
to puncture the cervical lymph-glands. 2 When these parasites are stained 
with a polychrome dye they show a rather large red nucleus about the mid- 
dle, a centrosome staining intensely in a vacuole-like area near the blunt 
posterior end, and a line of chromatin taking a dense red stain running down 




t|« J'^ 



Fig. 145. — Trypanosoma gambiense. {Da Costa.) 

the edge of the undulating membrane and terminating in the flagellum which 
is also stained red. The protoplasm of the body takes a distinct blue stain. 
The parasite contains no pigment and, therefore, obtains its nourishment 
from the plasma and not from the red cell. 

This disease may take an acute course, but as a rule is exceedingly chronic, 
running for years, but becoming fatal as soon as the parasite reaches the cere- 
brospinal fluid. The true sleeping sickness appears only when the cerebro- 
spinal fluid is invaded and seems to be, according to the recent work of Koch, 
directly amenable to treatment with atoxyl. In examining the cerebrospinal 
fluid for these parasites it is best gently to centrifuge the fluid for five minutes, 
after which the sediment may be examined under a vaselined cover-glass. 

There are many other types of trypanosomata,' but the Gambiense form 
is the more important. This is pathogenic toward man, but cannot be dis- 
tinguished from the trypanosoma of the tsetse fly which is so fatal to the 

1 See Thomson and Sinton (Ann. Trop. Med. and Parasitol., 1912, VI, 331), who have 
succeeded in cultivating this organism. Maynard, The Trypanosomes of Sleeping Sick- 
ness, 1915; Teichmann, Deutsche Med. Wchnschr., 1916, XLII, 1437; Adams, Jour. 
A. M. A, 1919, LXXIII, 1696. 

2 Wolbach and Binger (Jour. Med. Research, 191 2, XXVII, 83) show that the trypano- 
somes are not confined to the blood-vessels and lymphatics, but invade the tissues. See, 
also, Tuttle, Jour. Am. Med. Assn., 191 5, LXV, 240. 



634 DIAGNOSTIC METHODS 

horse and mule (trypanosoma Brucei), that of the surra disease^ (trypano- 
soma Evansi), or that of dourine (trypanosoma Equiperdum). 

(4) Kala-azar. 

Through the researches of Donovan, Leishman, and Ross, parasites have 
been demonstrated in the blood which are probably directly associated with 
the condition known as kala-azar, tropical splenomegaly, piroplasmosis, cach- 
exial fever, and dum-dum fever. The organism has been called the Leishman- 
Donovan body, and is a small oval, round, or oat-shaped body from 2j^ to 3 
microns in diameter. These bodies have a definite cell outline and contain 
two chromatin masses, a larger one, a nucleus which is almost round or oval 
and stains faintly, and a smaller bacillus-shaped centrosome which stains 
deeply and is directed almost at right angles to the axis of the nucleus. 
These two chromatin masses are both in the long axis of the cell, the larger one 
being at the periphery. Many of these forms are vacuolated and the outline 
of the cell cannot always be seen, although these two masses thus arranged are 
distinctive. They are easily stained with the various polychrome dyes and 
are best studied with the highest lenses. These bodies probably represent a 
stage in the development of a trypanosome as shown by the work of Leish- 
mann and Statham. They are not found in the circulating blood as a rule, 
but they have occasionally been reported in the form of intracellular bodies in 
fatal cases. They are easily seen in the blood obtained by splenic puncture 
and also in the granulation tissue taken from the ulcers. 2 Many are found 
in the mesenteric lymph-glands, bone-marrow, and liver. Some of these bodies 
lie free, but most of them are intracellular, either in the leucocytes, endothelial, 
or splenic cells, and frequently in large masses in the macrophages.^ 

The changes in the blood are those of a moderate anemia, associated with 
a leucopenia with a relative and absolute increase in the number of the large 
mononuclears. The average leucocyte count is about 2,000. 

(5) Filariasis. 

This is a condition associated with the presence of filariae in the blood 
{filaria sanguinis hominis). While many of these filariae^ are known; the 
most common one is the -filaria Bancrofti (filaria nocturna). These are from 
270 to 340 microns (0.2 to 0.3 mm.) long and from 7 to 11 microns broad. 

1 Mitzmain (Philippine Jour. Sc, B, 1913, VIII, 223) shows that this disease is trans- 
mitted by the tabanid fly {Tabanus Striatus Fabricus). 

2 Bates (Jour. Am. Med. Assn., 1913, LX, 898) reports a case of Leishmaniosis of the 
nasal mucosa, the organisms being obtained from direct smears made from the ulcers. See, 
also, Patton Indian Jour. Med. Research, 1914, II, 492; Mackie, Ibid., 510. 

3 Darling (Jour. Exper. Med., 1909, XI, 515) has reported the finding of an intracellular 
parasite, the histoplasma capsulatum, as the cause of Histoplasmosis, an infectious disease 
of the Canal Zone much resembling kala-azar. The parasite is small, round or oval, i to 
4m in diameter, possesses a polymorphous chromatin nucleus, basophilic cytoplasm and 
achromatic spaces all enclosed within an achromatic refractile capsule. 

^ Wellman and Johns (Jour. Am. Med. Assn., 191 2, LIX, 1531) have succeeded in culti- 
vating the filaria immitis on artificial media. See Fiilleborn, in Kolle and Wassermann's 
Handbuch der pathogen. Mikroorg., 1913, VIII, 185; Johns and Querens, Am. Jour. Trop. 
Dis. and Prev. Med., 1914, I, 620; Smith and Rivas, Ibid., 1914, II, 357; Dutcher and 
Whitmarsh, Ibid., i9i5;III, 65; Johnson, Southern Med. Jour., 1915, VIII,63o; Rosenberger, 
N. Y. Med. Jour., 1915, CII, 883; Lyon, Jour. A. M. A., 1917, LXVIII, 118; Yamada and 
Yamamoto, Mitt. a. d. med. Fak. Tokyo, 191 7, XVII, 87; Lynch, Jour. A. M. A., 1919, 
LXXVIII, 760. 



THE BLOOD 635 

They are enclosed in a sheath which is considerably longer than is the parasite 
and shows fine cross striations. The anterior end of the parasite is abruptly 
rounded and has a six-tipped prepuce and a sharp fang, while the posterior 
end tapers for about two-fifths of the length of the parasite. The median axis 
of the parasite is granular. The movement of these parasites is distinctly 
progressive at first as seen under the microscope, but they soon become 
motionless, appearing to attach themselves to the glass slide at their anterior 
end. Strangely enough these embryos appear in the circulation only toward 
evening, their numbers gradually rising to a maximum about midnight and 
diminishing toward dawn. During the day they are found in the internal 

® /^^^^ o 

Fig. 146. — Filaria Bancrofti. {Da Costa.) 

organs, especially the lungs. The forms appearing in the blood are prac- 
tically all embryos, as the adult types lie in the lymphatics where they ob- 
struct the lymph flow. 

The obstruction in the lymph-glands may also be brought about by the 
eggs, which are 25 to 38 microns long by- 15 broad. The embryos reach the 
general circulation only through the thoracic duct. The female filaria is 85 
to 150 mm. long with a distinct neck, a head with a simple, minute, terminal 
mouth, and a plain cylindrical body covered by a striated cuticle and tapering 
toward the neck and tail. The tail ends bluntly and has a small depression 
surrounded by two lips. The male is about 80 mm. long, without a neck and 
having a tendril-like tail rolled into one or more spirals. 

Like the malarial organisms, the filaria has an intermediate host in the 
mosquito, both of the culex and anopheles variety. The embryos, which 
are taken up by the bite of the mosquito, cast off their sheath in about one 
hour in the stomach of a mosquito. Some of these embryos die at this stage, 
but others bore actively through the intestinal wall to the muscle, where they 
remain. In the next two or three days the embryo becomes larger and its 



^ 636 DIAGNOSTIC METHODS 

alimentary tract develops. On the seventh day the worm is about i3^ 
mm, long and is perfectly developed. It now travels toward the head and 
takes its position in the labium, whence it enters the blood of its new host 
during the biting by the insect. A large number of these adult forms is neces- 
sary to cause very severe cases and many years pass before any symptoms 
are manifest. 

In examining the blood for the filaria, it is best to take a specimen late 
at night and to make a very thick, fresh specimen which should be examined 
with a low pov/er.^ Besides the ordinary anemia which may develop in such 
cases, we find a very striking eosinophilia which may run from 4 to 17 per 
cent. 

A very characteristic finding in such cases is the condition of hematochy- 
luria followed by chyluria. This hematochlyuria seems to be due to rupture 
of the varicose lymph-vessels of the bladder, as these form a large part of the 
i collateral circulation when the thoracic duct is occluded (Emerson). Such 

' attacks may occur for years and be separated by long intervals. Their onset 

is spontaneous or following exertion and is usually associated with pain and 
fever. The urine shows the presence of blood, chyle (as high as 3.8 per cent. 
fat), and embryos. 

Many other forms of filariae are known, but this Bancroft type seems to 
be the more important. While this disease occurs endemically in the tropics 
there are undoubtedly many cases in this country. It is, therefore, wise in 
a case showing lymph tumor, elephantiasis, and hematochyluria, especially 
when pain and fever and enlarged spleen are present, to examine the blood 
for the filaria Bancrofti. 

(6) Syphilis. 
1 The search for the etiologic factor of syphilis has extended over a period 

of many years and various organisms have been described, from time to lime. 
I However, none of these former parasites have stood the test of criticism. 

\ Through the work of Schaudinn and Hoffmann in 1905, a parasite was dis- 

i' covered which was so constantly associated with syphilitic lesions, whether 

•primary, secondary or tertiary, that it was almost universally regarded as the 
[ causative agent in this disease. The cultivation of this organism in pure 

'' culture by Noguchi (see p. 639) and the demonstration of the pathogenic 

: properties of this cultivated parasite have definitely established the etiologic 

i relationship of this organism to syphilis. This organism was called the 

spirochete pallida or treponema pallidum. 
5 While it is now a relatively simple procedure and a matter of almost daily 

I laboratory routine to demonstrate the presence of these organisms in the pri- 

l mary and secondary syphilitic lesions or in the blood of affected patients, 

' the detection of the spirochete in the tertiary stages or in the parasyphilitic 

I conditions is not by any means always successful. It is in the latter types 

I of this disease that the Wassermann and luetin tests are so frequently called 

j ^ The concentration method of Smith and Rivas is of advantage. This consists of add- 

ing I c.c. of blood to 10 c.c. of 2 per cent, acetic acid, centrifuging, washing and centrifuging 
several times, spreading the sediment on a slide and examining with low power lens. 



THE BLOOD 637 

upon to settle the diagnosis, as the primary and secondary stages are more 
amenable to direct diagnosis. 

It has, of course, been known for some time that chronic nervous condi- 
tions, such as general paralysis and tabes dorsalis, were closely associated with 
long-standing syphilitic infection, but this relationship remained to be clearly 
proven. Noguchi and Moore^ have demonstrated the presence of the tre- 
ponema in the brain tissue of paretics and also in the posterior columns of the 
spinal cord in tabes. These results have been abundantly confirmed by 
others. It is true that the results show only about 25 per cent, of positive 
findings in these cases but this is not surprising in view of the latent character 
of the infection. Such a result indicates, merely, the association of these 
organisms with the pathologic lesions of the disease and does not prove their 
absolute etiologic relationship to syphilis in general. Later work byNoguchi 
and others has clearly shown that typical syphilitic lesions may be caused in 
rabbits by inoculation of an emulsion of the brain of a paretic individual. 
The results show about the same percentage of successful inoculations as do 
those obtained from direct histologic examination of the tissues.^ Graves^ 
goes even a step further and believes that his experiments show that rabbits 
may be infected with syphilis directly from the blood of general paretics. 
The proof is, therefore, absolute that these diseases, general paralysis and 
tabes dorsalis, are the result of actual syphilitic infection of the brain and 
spinal cord. It is evident, therefore, that the Wassermann and luetin tests 
can be of inestimable service in such cases in the way of clearing up a diagnosis. 

The spirocheta pallida^ {treponema pallidum) derives its name from its 
low refractive power and the difficulty with which it takes up anilin dyes. It 
has a very delicate structure, usually presenting 10 to 40 deep spiral incurva- 
tions in the larger specimens or only a few in the smaller ones. Its length 
varies between 4 to 10 microns and its width does not exceed J^ micron. 
Noguchi has divided the strains of this organism into a thick, a thin and a 
medium type. The organism has been demonstrated in the circulating blood, 
in the scrapings obtained from the chancre, in the incised papules, in smears 
from the mucous patches, and in the fluid aspirated from the inguinal glands. 
It seems to be easily demonstrable in the blood from a splenic puncture, while 
in the congenital forms it is found in the internal organs and in the peripheral 
blood. A characteristic difference between this spirocheta and some other 
types (spirocheta buccalis), with which it might be confused, is that its 

^ Jour. Exper. Med., 1913, XVII, 232; Noguchi, Jour. Am. Med. Assn., 1913, LXI, 85; 
Miiiich med. Wchnschr., i9i3,LX, 737; Jour. Cutan. Dis., 1913, XXXI, 543; Forster and 
Tomasczewski, Deutsch. med. Wchnschr., 1914, XL, 694; Warthin, Am. Jour. Med. Sc, 
1916, CLII, 508; Keil}^, Jour. Lab. and Clin. Med., 191 7, II, 260. 

2 See Hoche, Med. Klin., 1913, IX, 1065; Forster and Tomasczewski, Deutsch. med. 
Wchnschr., 1913, XXXIX, 1237; Wassermann, Ibid., 1281; Levaditi, Marie and Bankowski, 
Ann. de I'lnst. Pasteur, 1913, XXVII, 577; Nichols and Hough, Jour. Am. Med. Assn., 
1913, LXI, 120; Wile, Ibid., 866; Wile and De Kruif, Jour. A. M. A., i9i6,LXVI, 646; 
Reasoner, Ibid., 1916, LXVII, 1799; Wile, Jour. Exper. Med., 1916, XXIII, 199; Grannelli, 
Policlinico, 1917, XXIV, 3. 

^ Jour. Am. Med. Assn., 1913, LXI, 1504. 

''See McDonagh, Brit. Jour. Dermatol., 191 2, XXIV, 381; and Ross, Lancet, 191 2, II, 
1 105. See Noguchi, Spirochetes (Harvey Lecture), Jour. Lab. and Clin Med., 1917, II, 
365 and 472; Am. Jour. Syph., 1917, I, 261. 



638 



DIAGNOSTIC METHODS 



f • 



ends lie above and below a longitudinal line drawn through the center of its 
curvatures, while in the other forms the ends lie on the projection of such a 
line.^ The organism moves in an oscillatory manner about its longitudinal 
axis, its movements being winding, bending and whipping, while in the 
spirilla the longitudinal axis remains rigid. 2 Schaudinn demonstrated the 
existence of a flagellum at each end, while the other spirochetae have an 
undulating membrane. 




Fig. 147. 



-Spirochetae pallidae and refringens. 
The darker ones are the refringens. 



(Pitfield.) 



These organisms are seen only with great difficulty in the specimens of 
fresh blood, but thanks to the introduction of the ultra-condenser (the dark- 
field illuminator) we are in a position to see these organisms, both in the 
splenic and peripheral blood, although considerable practice is necessary prop- 
erly to adjust the light. ^ These organisms do not take anilin dyes readily, so 
special methods have been advanced for their demonstration in smears. 
A very good stain for them is the Goldhorn stain. The smears are fixed 

^ Ciarla (Policlinico., 191 7, XXIV, 369) calls attention to the fact that the spirals are 
extremely regular and appear to be coiled around a central axial filament whose demon- 
stration is doubtful. 

2 Zinsser and Hopkins (Jour. A. M. A., 1914, LXII, 1892) have shown that the trepo- 
nema may live and remain motile 11 /i hours on wet towels exposed to room temperature. 
Reasoner (Ibid., 1917, LXVIII, 973) shows that soap, as applied in shaving, destroys the 
motility of these organisms at once. The importance of these observations as to possi- 
bility of transmission of syphilis by barbers is evident. 

^ See Sid well and Smith, 111. Med. Jour., 19 14, XXVI, 418. See, Special Report Series 
No. 19, of Med. Res. Com. National Health Insurance, London, 1918, for a full discussion 
of the principles of dark field illumination. 



THE BLOOD 



639 



with pure methyl alcohol for 15 minutes and then covered with the stain 
(polychrome methylene blue) for three to five seconds, when the excess is 
drained off. .The specimens are then slowly introduced into clean water 
with the film sides down. Keep the slide in this position for four to five 
seconds and then shake in the water to remove the excess of the dye. The 
spirochetae appear of a violet color. This violet tint may be changed to a 
bluish-black by covering the specimen with Gram's iodin solution for 15 to 
20 seconds, after which it is washed and dried as usual and the examination 
made with the immersion lens. The writer has also found the use of the 
Giemsa stain very reliable, especially when the staining is continued for 18 
hours (see Exudates). Other stains, such as that of Levaditi, have been advo- 
cated, but they do not seem to give any better results and are more 




Fig. 148. — Ultra-condenser of Reichert. 



complicated. For staining the spirochetae in tissues the Levaditi stain is 
admirable. 

The examination of the blood is very often disappointing, owing to the 
fact that few spirochetae may be present in the specimen. Better results 
are obtained by examination of specimens from a curettage which has been 
carried sufficiently far to allow serum to appear. This serous fluid is then 
spread upon slides and treated in the usual manner (see Exudates). 

Cultivation of Treponema Pallidum. 

As previously stated, the causative factor of syphilis has been definitely 
settled only by fulfilling Koch's postulates regarding pure cultures and the 
production of the disease by means of these pure cultures. Schereschewsky,^ 
Miihlens^ and Hoffmann^ were able to cultivate the pallidum but were 

^Deutsch. med. Wchnschr., 1909, XXXV, 835, 1260 and 1652; Ibid., 1913, XXXIX, 
1408. 

^Deutsch. med. Wchnschr., 1909, XXXV, 1261; Klin. Jahrb., 1910, XXIII, 339. 
3Ztschr. f. Hyg. u. Infectkr., 1911, LXVIII, 27. 



640 DIAGNOSTIC METHODS 

unable to reproduce syphilitic lesions by means of their cultures. Bruckner 
and Galasesco^ and Sowada^ reported the successful reproduction of the 
lesions by injection of their ''young impure cultures" but, as neither investi- 
gator was able to grow a second generation of these so-called cultures in 
any medium, their results are questionable. Recently Noguchi^ has suc- 
ceeded not only in producing many generations of pure cultures of the 
pallidum but, also, in reproducing the syphilitic lesions by use of these pure 
cultures. His work is of the greatest importance as it absolutely establishes 
the spirocheta pallida as the etiologic factor of syphilis. 

His technic is as follows: As the material for obtaining the cultures 
he uses the spirochetae-containing testicular tissue of rabbits, which have been 
inoculated with human syphilitic material. From this first generation in 
rabbits any number of generations may be derived by transmitting the strain, 
at appropriate intervals (four to six weeks), from rabbit to rabbit. By this 
means the spirochetae are almost free from banal bacteria. 

The culture media is the following: 15 c.c. of a mixture of equal parts of 
ascitic fluid and bouillon are placed in tubes 20 cm. long and 1.5 cm. wide. 
These tubes are then sterilized by fractional sterilization at 100 C. for 
15 minutes on each of three successive days. Then a small piece of freshly 
removed sterile rabbit tissue (preferably kidney or testicle, although heart 
muscle may be used but not liver) is placed in each tube which is then 
incubated at 37°C. for two days and examined for sterility To each tube 
a layer of sterile paraffin oil is added to shield the medium from contact 
with air and to prevent evaporation. The method of cultivation must 
be strictly anaerobic in obtaining the first generation of pallida. Noguchi 
employs a combination of hydrogen gas, vacuum and pyrogallic acid in an 
anaerobic apparatus, which is fully described in his article. He believes 
the following conditions, essential in obtaining his first generation: (i) the 
presence of suitable fresh sterile tissue in serum- water, (2) strict anaerobiosis, 
(3) a slightly alkaline reaction as furnished by the serum and tissue, and (4) 
a temperature of about 35 to 37°C. 

When once adapted to the artificial ascitic-broth tissue medium, the 
pallidum grows well under less strictly anaerobic conditions. In fact, certain 
strains even grow well in a medium containing agar, provided suitable fresh 
tissue is placed low down in a high column of medium (serum- water or serum 
agar), and is covered with paraffin oil. Out of ten strains of treponema pal- 
lidum, six have been cultivated in the above medium. The difficulty in this 
work seems to be to obtain the first generation. The culture may be purified 
by permitting it to grow through a Berkefeld filter, which the pallida pass 
about the fifth day. Certain strains grow together with bacteria along the 
stab canal in a serum-agar tissue medium. But while the bacteria do not 

^ Compt. rend. Soc. de biol., 1910, LXVIII, 684. 

2Deutsch. med. Wchnschr., 1911, XXXVII, 682; Med., Klin., 1914, X, 161. 

3 Jour. Exper. Med., 191 1, XIV, 99; Ibid., 191 2, XV, 90; Ibid., 201; Ibid., 191 2, XVI, 
211. See, also, Nichols, Ibid., 1914, XIX, 362; Hartwell (Jour. Am. Med. Assn., 1914, 
LXIII, 142) reports the isolation of this organism from the blood. Zinsser, Hopkins 
and Gilbert, Jour. Exper. Med., 1915, XXI, 213; Akatsu, Ibid., 191 7, XXV, 375; Noguchi 
and Akatsu, Ibid., 765. 



THE BLOOD 641 

grow out into the surrounding medium, the pallida grow out gradually in a 
pure state. The characteristics of this pure culture may be obtained from the 
literature.^ 

The morphology and motility are quite typical, difficulty being experi- 
enced in distinguishing these points from those of specimens taken from 
human lesions. These pure cultures produce typical lesions when injected 
into animals, thus completing the chain of evidence. ^ 
(7) Yellow Fever. 

This infectious noncontagious disease is caused by a specific organism 
recently discovered by Noguchi and named the Leptospiraicteroides . That 
the disease was blood-borne was known from the fact that injection of blood 
from a yellow-fever patient into healthy subjects caused the disease. Fur- 
ther, the organism was known to be capable of passage through a Berkfeld 
filter. The various organisms, which have, in times past, been held respon- 
sible for this disease, among them the Bacillus X of Sternberg, the Bacillus 
icteroides of Sanarelli and the myxococcidium stegomyiae of the Yellow Fever 
Commission, must now be abandoned in view of the work of Noguchi and his 
associates. 

In 1881 Finlay advanced the hypothesis that yellow fever was transmitted 
to man only through the bite of a mosquito of the Culex group, the stegomyia 
fasciata. The United States Commission, consisting of Reed, Carroll, Agra- 
monte, and Lazear, furnished the experimental proof that this hypothesis was 
valid and showed that the unknown organism of the disease required a period 
of 1 2 days' development in the body of the mosquito before it could be trans- 
mitted from the stegomyia as an infecting agent. A second U. S. Commission, 
consisting of Parker, Pothier, and Beyer with the help of Smith stated in 1903 
that yellow fever was due to a parasite of the sporozoan type, the myxococ- 
cidium stegomyiae, which developed in the stegomyia. This organism has 
never been found in the human body, hence its schizogony (asexual develop- 
ment) is unknown. The French Commission, Marchoux, Salimbeni, and 
Simond, as well as Schaudinn and Carroll, do not believe that this organism 
has anything to do with yellow fever. ^ Seidelin^ has reported the finding 

1 See Meirowsky ("Studien iiber die Fortpflanzung von Bakterien, Spirillen und Spiro- 
chaten," Berlin, 1914) for a discussion of the proper classification of this organism. 

2 In his later work Noguchi has cultivated the organism directly from the lesions in man, 
using a high cylindrical layer of solid media consisting of two parts of 2 per cent, slightly 
alkaline agar and one part of ascitic or hydrocele fluid, at the bottom of which has been 
placed a fragment of sterile tissue. The small pieces of tissue taken from the lesion are 
inserted deep into this media and the growth carried on as outlined above. See, also, 
Baeslack, Jour. Infect. Dis., 1913, XII, 55. Noguchi has, also, cultivated various types of 
spirochetae, which closely resemble the pallida, and has thus shown their morphological 
variations (Jour. Exper. Med., 1912, XV, 466; i9i2,XVI, 194, 199, 261 and 620; Ibid., 1913, 
XVII, 89). See also, Clark and Gates, Am. Jour. Dis. Child., 1915, IX, 126; Zinsser 
Hopkins and Gilbert, Jour. Exper. Med., 1915, XXI, 213. Zinsser and Hopkins (Ibid., 
576) as, also, Kissmeyer (Deutsch. med. Wchnschr., 191 5, XLI, 306) have shown the pres- 
ence of anti-bodies (agglutinins) for the treponema pallidum in the blood of syphilitics. See 
Zinsser and Hopkins, Jour. Exper. Med., 1916, XXIII, 323; Zinsser, Hopkins and McBurney, 
Ibid., 329 and 341; Ibid., XXIV, 561; Kolmer, Broadwell and Matsunami, Ibid., t^t^^)- 

^ See Yellow Fever, Senate Document No. 822, Washington, 191 1. 

* Jour. Path, and BacterioL, 191 1, XV, 282; Otto, inKolle and Wassermann's Handbuch 
der pathogen; Mikroorg., 1913, VIII, 623. See, also, Wenyon and Low, Jour. Trop. 
Med. and Hyg., 1914, XVII, 369; Ibid., 1915, XVIII, 55; Camerer, U. S. Naval Med. Bull, 
1915, IX, 65. 



642 DIAGNOSTIC METHODS 

of a parasite, a protozoon, in the blood, which he believes the causative 
factor. 

In 1 9 18 the Yellow Fever Commission of the International Health Board, 
consisting of Drs. A. I. Kendall and Charles A. Elliott and Mr. H. E. Reden- 
baugh of Northwestern University Medical School, Chicago, 111. ; Dr. Mario 
Lebredo of Las Animas Hospital, Havana, Cuba; and Dr. Hideyo Noguchi of 
the Rockefeller Institute for Medical Research, New York, undertook the 
study of this disease in Guayaquil. The work of this Commission and especi- 
ally of Noguchi^ has demonstrated that the etiologic factor in this disease is 
the Leptospira icteroides, an organism differing from both the spironema 
and treponema but closely related to the organism of Weil's disease the 
Leptospira icterohemorrhagica) . This organism has been recovered from the 
blood of infected patients, has been transmitted direct from patients to 
animals (guinea pigs), has been found in the blood of infected animals and 
obtained in pure culture. The further link in the chain has been recently 
published by Noguchi in the transmission of the infective agent from an 
infected patient, through the bite of the stegomyia calopus, to animals, which 
animals manifested the symptoms of the disease and from which the organism 
was obtained and cultured. 

This organism, which occurs in the blood and tissues in yellow fever 
patients, as well as in those animal experimentally infected with the blood or 
tissue of yellow fever patients, is an extremely delicate filament, measuring 
about 4 to 9 microns in length and 0.2 of a micron in width along the middle 
portion. It tapers gradually toward the extremities, which end in immeas- 
urably sharp points. The entire filament is not smooth but is minutely 
wound at short and regular intervals, the length of each section measuring 
about 0.25 of a micron. The windings are so placed as to form a zigzag line 
by the alternate change of direction of each consecutive portion at an angle 
of 90 degrees. It is unrecognizable by translucent light but becomes quite 
visible under dark-field illumination. It possesses an active motility, con- 
sisting in vibration, rotation, rapid bipolar progression, and sometimes 
twisting of parts of the filament. When it encounters a semisolid substance 
it penetrates the latter by a boring motion, and while passing through it the 
body assumes a serpentine aspect. It manifests remarkable flexibility to 
almost any angle while changing its course of progression in a semisolid 
medium. In a fluid medium, one end is usually bent in the form of a graceful 
hook, and, while rapidly rotating, the organism proceeds in the direction of 
the straight end, the hooked end apparently serving as a sort of propeller. 
Many specimens are seen with both ends hooked, the organism then rotating 
in a stationary position unless one hook is larger and more powerful. The 
rapid rotation mak^s the organism appear like a chain of minute dots. 

This organism is difficult to stain with ordinary aniline dyes, but can be 
made distinct by osmic acid fixation and staining with Giemsa or Wright 
stain. When stained with Fontana's method (see Exudates) the organism 

1 Jour. Exper. Med., 1919, XXIX, 549, 565, and 585; Ibid., Ibid., XXX, i, 9, 13, 87, 
95, and 401; Jour. A. M. A., 1919, LXXII, 187; Lebredo, Vida Nueva, 1919, XI, 145. 



THE BLOOD 643 

appears as a moderately heavy, slightly undulated filament without a clear 
elementary indentation. The peculiar forms resembling the letters C and 
S are quite characteristic. Specimens fixed with methyl alcohol seldom 
retain the elementary spirals. From these findings it is evident that the 
present organism falls in the general order of so called spirochetes, but in 
the strict sense of the term it is neither a bacterium, a spirochete, a spiro- 
nema, nor a treponema, but belongs to the genus Leptospira. For methods 
of cultivation see the original literature. 

As this disease is beyond question blood-borne, its hematological changes 
are of some interest. Jones shows that anemia is infrequent, that fibrin 
formation is deficient, that the globucidal action of the serum is greatly 
increased, and that both cholemia and hemaglobinemia occur. The red cells 
show little variation in number, Pothier never finding them below 4,280,000. 
The hemoglobin suffers considerable loss, being usually between 50 and 75 
per cent. This loss is restored very slowly in convalescence. Albertoni 
draws attention to the lack of parallelism between the percentage of hemo- 
globin and the specific gravity of the blood, the latter falling much more 
than the former. Morphological changes in the reds are rare. An occasional 
normoblast may be seen. 

The leucocytes range between 4,660 and 20,000, the higher the count 
the more favorable the prognosis. In this leucocytosis the polynuclear 
neutrophiles are in higher proportion, only rarely being normal.^ Eosinophiles 
are few and myelocytes only occasional. 

(8) Infectious Jaundice. — ^Weil^s Disease. 

Although infectious jaundice had been known for some time to exist 
among troops, sewer workers, miners, and rice planters (especially in Japan), 
it remained for Weil in 1886 to describe this entity in such a way that the 
disease is commonly known, today, as Weil's disease. It is characterized 
.by sudden onset with malaise, chills, high fever, intense muscular pains, 
jaundice and evidences of acute nephritis, such as the appearance of albumin 
and casts in the urine; severe cases may be accompanied by epistaxis, skin 
and submucous hemorrhages, and lymphadenitis. 

This disease prevails rather extensively in Japan, so that opportunity for 
its study there was excellent. In 1914, Inada, Ido, Hoki, Kaneko, and Ito 
transmitted this disease to guinea pigs by inoculating them with the blood 
of patients suffering from the disease.^ They were, further, able to discover 
in the blood and various organs of the experimental animals, a new spiro- 
chete, to which they gave the name Spirocheta icterohcemorrhagica and which 
is now established as the causative agent of this disease. Some time later 
Uhlenhuth and Fromme^ and, independently, Hiibener and Reiter* accom- 

^ Noguchi states that the number of leucoytes soon returns to normal and in a few days 
a marked leucopenia sets in. 

2 Jour. Exper. Med., 1916, XXIII, 377. See, also, Ito and Matsuzaki, Ibid., 557; 
Ido, Hoki, Ito, and Wani, Ibid., 1916, XXIV, 471; Inada, Ryokichi, Ido, Yutaki, Hoki, 
Rokuro, Ito, Hiroshi, and Wani, Ibid., 485. 

^ Med. Klin., 1915, XI, 1202, 1264, 1296, and 1375. 

^Deutsch. Med. Wchnschr., 1915, XLI, 1275; Ibid., 1916, XLII, i; See, also, Reiter, 
Ibid., 1283. 



II 



644 



DIAGNOSTIC METHODS 



i< ' 



plished the same thing, the latter workers nammg their organism, Spirocheta 
nodosa. Still later Stokes and Ryle^ succeeded in transmitting the disease to 
guinea pigs by inoculating them with blood from the infected British soldiers 
on the Flanders' front, but Stokes, Ryle, and Tytler^ did not decide the 
question of the identity of their organism with that of Inada and his asso- 
ciates. Many workers on the French front demonstrated the presence of 
these spirochete in their cases and regarded them as closely related to the 
Japanese type.^ On the Italian front Monti^ has arrived at the same con- 
clusion. According to Noguchi, "In America, especially the United States, 
there have been few epidemic or endemic cases of infectious jaundice re- 
ported from various quarters of the continent (Toronto, Middle Western and 
Southern United States) and from Cuba, but it was not known whether or 
not these cases corresponded with those found in Europe and Asia." As 
this disease appears to be widespread, especially in times of war, it is important 
that its etiology be understood and capable of recognition. The work of 
Noguchi has shown that these various strains are probably identical. 

Although Inada and his coworkers first isolated, cultivated, described 
and named the etiologic factor of Weil's disease, yet the description of 
Noguchi, based on his studies, is so much fuller, that the following is quoted 
from his discussion of the subject.^ The elementary structure of this 
organism is that of a closely wound slender cylindrical thread with gradually 
tapering ends, averaging 9 by 0.25 micron. Individuals of 3 to 4ju or 20, 30 
and even 4o,u are met with in culture. The number of coils is greater in a 
given length that of any spirochete studied. It is so closely wound that with- 
in 5Ai there are 10 to 12 coils. Near the extremities the coils become closer. 
They are never very deep, the aspect of the whole body being that of a trans- 
versely barred chain of streptococci The winding is rarely seen distinctly, 
although it can be brought out well by a carefully fixed stained preparation 
(see Exudates) or under powerful dark-field illumination. The movement 
and customary position of the organism in free space are characteristic. 
Active specimens show a straight body with one or both ends curved in the 
form of a semi-circle. The length of the hook at the end varies somewhat, 
but is usually about 3 to 5iu. While in motion, the organism, without re- 
laxing its elementary minute windings, rotates around its axis, making about 
two to four turns per second, giving the impression of a drawn out figure eight. 
The movement is bipolar, and its direction alternates at short intervals. 
When passing through a semisolid medium, the body of the spirochete 
assumes a wavy spiral, the movements being brusque and erratic. There 

^ Jour. Royal Army Med. Corps, 1916, XXVI, 286. 

2 Lancet, 1917, I, 142. 

^ Martin and Pettit, Presse Med., 1916, XXIV, 569; Bull, de I'Acad. Med.. 1916, 
LXXVI, 247; Costa and Troisier, C. R. soc. biol. Paris, 1916, LXXIX, 1038; Bull, et 
mem. Hop. Paris, 1916, XL, 1928; Merklen and Lioust, Ibid., 1865; Garnier and Reilly, 
Ibid., 2249; Ameuille, Ibid., 2281; Renaux, C. R. soc. biol. Paris, 1916, LXXIX, 947; 
Salomon and Xeveu, Ibid., 191 7, LXXX, 272. 

'' Boll. Soc. med. chir. Pavia, 1916; Policlinico, 191 7, XXIV, 290; Moreschi, Ibid., 265; 
Micheli and Satta, Ibid, .268; Trincas, Ibid., 271; Pontano, Ibid., 277; Merighi, Ibid., 280. 

^ Jour. Exper. Med., 1917, XXV, 755; Ibid!, 1918, XXVII, 575, 593, and 609. See 
Otteraaen, Jour. Infect. Dis., 1919, XXIV, 485. 



THE BLOOD 



645 



is a distinct halo around the organism, but no membrane has been demon- 
strated. No minute flagellum-like projection could be demonstrated by 
staining. • The hooked ends form one of the most characteristic poses of the 
organism while rotating on its axis in a free space, but as soon as it meets a 
solid or semi-solid obstacle, it begins to penetrate into it. It is devoid of a 
terminal filament such as is characteristic of a spironema or treponema, and 
is resistant to 10 per cent, saponin solution, unlike all other known spiro- 
chete. For these reasons, Noguchi 
believes it should be called Lep- 
tospira icterohcemorrhagica. 

The finding by- Noguchi of 
this organism in American wild 
rats and his identification of it 
with the strains observed in Asia 
and Europe seem to indicate the 
source of infection in these cases. 
As the rats examined carried this 
organism in their kidneys and as 
the urine of infected animals, as 
well as of men, contains large 
numbers of these spirochete, it is 
reasonable to infer that infection 
may arise from the contaminated 
soil, the organism gaining en- 
trance into the body through the 
feet. This is especially prone to 
occur with the bare-footed workers 
in rice fields in Japan and with 
soldiers, whose shoes have been 
worn through by the exigencies 
of the campaign. 

(9) Rocky Mountain Spotted 
Fever (Tick Fever). ^ ^ ,. ^ ■. 

. riG. 149. — Schistosomum nematobmm; male 

This disease is not to be con- with female in gynecophoric groove. {Tyson 
fused with typhus fever or epidemic ^^^^^ Loos.) 
cerebro-spinal meningitis, to both 
of which the term "spotted fever" has been occasionally applied. 

Wilson and Chowning have reported that the blood of man affected 
with Rocky Mountain spotted fever shows the presence of an erythrocytic 
parasite which they call Piroplasma hominis. These parasites are ovoid in 
form, have ameboid motility, and are unpigmented Three forms of these 
intracellular ovoids were found: (i) a small, non-motile form, i to 2 
microns in length by i micron in width; (2) a larger actively ameboid form, 
3 to 5 microns in length by i to 1.5 microns in width, and showing a dark 
granular spot at one end; (3) a twin form, consisting of two pear-shaped 




T I 



646 



DIAGNOSTIC METHODS 



bodies, lying with their tapered ends approaching and bearing a granular 
spot at each end. These bodies stain best with the polychrome dyes. 

Through the work of Ricketts and King it has been definitely established 
that the parasite of spotted fever finds its host in the wood tick (dermacentor 
venustus (Banks) or, as Stiles states, the dermacentor andersoni). There is 
apparently no cycle of development in the tick as an intermediate host. 
Ricketts advances much evidence against the piroplasma as the causative fac- 
tor, but is unable to say that such might not be the etiologic unit. After 
much research he^ succeeded in finding an extra- and intra-cellular pleomor- 
phic polar staining bacillus, which is extremely small and is constantly present 
in the blood of infected animals as well as in the infecting tick. Whether 
this is the true etiologic factor is still unsettled, but Ricketts believed it was. 2 

The red cells in this disease are reduced to about 4,000,000, while the 
hemoglobin content may be as low as 50, thus giving a low color index. 
Degenerations and atypical staining qualities are rare. The leucocytes are 
increased to 12,000 or more and show nothing differentially abnormal, except 
a slight increase of the large lymphocytes. 
(10) Distomiasis (Bilharziasis). 

This is a chronic parasitic disease due to the deposition in the tissues of 
the eggs of the worm, Schistosomum hematobium, also called Bilharzia hema- 
tobia, gynecophorus, distomum hematobium, distoma capense, and Thecos- 
oma. It is a very common condition in Africa, but has been found but six 
times in America, according to O'Neil. Infection appears to be more com- 
monly induced by drinking unfiltered infected water, but occasionally it 
may come through the skin.^ 

The adult parasites inhabit the blood of the portal vein and the vessels 
of the pelvis, rectum, and bladder. The male is smaller and thicker than the 
female, is 12 to 15 mm. long and i mm. broad, is flat and so folded as to form 
a gynecophoric canal which holds the female. The female is about 20 mm. 
long and 0.25 mm. thick and is the active agent in the infection. 

The eggs of the parasite are oval or spindle-shaped, measure about 0.16 
mm. in length and 0.05 mm. in breadth, and have a distinct spine-like pro- 
jection from the posterior end or from one side. These ova are particularly 
frequent in the urine of such cases. Occasionally they may be found in the 
circulating blood. 

VI. Bacteriology of the Blood 



From the standpoint of differential diagnosis and treatment of a con- 
dition known or thought to be of infectious origin, it is frequently of especial 

^ Jour. Am. Med. Assn., 1909, LII, 379. 

2 See McClintic (Pub. Health Rep., 191 2, XXVII, 732) for an excellent bibliography of 
the subject; also, Rucker, Ibid., 191 2, XXVII, 1465; Wolbach (Jour. Med. Res., 1916, 
XXXIV, 121; XXXV, 147; and 191 8, XXXVII, 499; 1919, XLI, i) confirms the findings 
of Ricketts. See, also, Michie and Parsons, Med. Record., 1916, LXXXIX, 265. 

^ SeeLooss, in Kolle and Wassermann's Handbuch d. pathogen. Mikroorg., 1913, VIII, 
i; Kartulis, Ibid., 23; Bovaird and Cecil (Am. Jour. Med. Sc, 1914, CXLVIII, 187) report 
cases of infection with the closely related schistosomun japonicum; Reed, Am. Jour. Trop. 
Dis. and Prev. Med., 191 5, III, 247; Mann, Jour. A. M. a\., 1916, LXVII, 1366; Ferguson, 
Jour. Royal Army Med. Corps, 191 7, XXIX, 57. 



THE BLOOD 647 

importance carefully to examine the blood for the presence of various bacteria. 
Moreover, in many conditions, as for instance in typhoid fever, cultures made 
from the circulating blood often give positive findings long before other tests 
are obtainable. The bacteriological study of the blood requires not only a 
thorough working-knowledge of the principles underlying the bacteriological 
technic but, in many cases, special training, also, in the isolation and differ- 
entiation of the suspected organism.^ 
Technic. 

The success of this work depends partly upon the obtaining of a suflS- 
cient quantity of blood and partly upon the selection of the culture-media for 
the future development of the organism present. The amount of blood re- 
quired varies from 5 to 20 c.c, so that it must be obtained by venous puncture. 
The vein usually selected is the median basilic or the median cephalic at the 
bend of the elbow, but, if these be not available, the veins of the dorsum of the 
hand may be used, although the latter are less satisfactory. The site of 
puncture should be carefully cleansed, using all the precautions taken for any 
surgical procedure. As a rule, contaminations from the skin are rare pro- 
viding this cleansing has been adequate. In some cases, especially in very 
obese patients, it is necessary to expose the vein by incision of the skin and 
subcutaneous fat over the vein. 

The instrument best adapted for venous puncture is, in the writer's opin- 
ion the Llier syringe, which is made entirely of glass and has a tightly fitting 
platinum needle. ^ Sterilization is best done by the use of dry heat to i5o°C. 
for one hour, boiling in i per cent, sodium carbonate solution for 45 minutes, 
or using steam under pressure in an autoclave for 15 minutes. These instru- 
ments should never be sterilized with carbolic acid or bichlorid solution owing 
to the danger of inhibiting the growth of the suspected organisms. It is 
well, also, to sterilize a pair of forceps to use in fitting the needle on the syringe. 
The properly sterilized outfits should be kept in long test-tubes, plugged with 
cotton, the point of the needle being protected by resting upon a pad of 
steriHzed cotton. 

When the above precautions have been taken, an elastic band or a towel 
may be fastened about the arm above the point of puncture, to produce con- 
striction of the vessels and distention at the point of puncture. The skin 
may be anesthetized with a spray of ethyl chlorid, if the patient be very sus- 
ceptible to the sUght pain caused by the puncture. The needle is plunged 
into the vein against the direction of the blood current, care being taken that 
the point of the needle is sharp as, otherwise, the vein may roll about beneath 
it. Very slight aspiration is necessary to draw the blood into the syringe, as 
the blood tends to flow into the instrument through its own force. The usual 
amount withdrawn is 10 c.c. but occasionally 20 c.c. are preferable, especially 
if the patient is plethoric and can stand the loss easily. When the desired 
amount is obtained, the bandage is removed before the needle is withdrawn. 

^ See Cummer, Ohio State Med. Jour., 1914, X, 201; Lintz, New York Med. Jour., 1915, 
CI, 660; McLeod and Bevan-Brown, Jour. Path, and Bact., 1918, XXII, 76. 

2 See Judd and Simon (Jour. Am. Med. Assn., 1915, LXIV, 822) for the use of the Keidel 
tube charged with culture medium. 



648 



DIAGNOSTIC METHODS 



W 



After withdrawal from the vein, the needle is quickly removed from the 
syringe and the blood divided among several tubes containing agar melted 
and cooled to 40°C., the tip of the syringe being passed through the flame 
before inoculating each tube. In deciding as to the amount of blood to add 
to the agar, a general rule is to increase the amount of blood when the feeble- 
growing organisms, such as the pneumococcus and gonococcus, are sus- 
pected. The usual proportions are 2 to 3 of blood to 5 of agar. It is, 
probably, preferable to add the blood to fluid media, such as bouillon or 
litmus-milk, using i to 2 c.c. of blood to 100 c.c. of media in order to over- 
come the normal bactericidal effects of the blood.^ The blood and fluid 
media are then directly incubated at 37°C. In the case of the agar tubes, 
the blood and media are mixed by shaking as quickly as possible, poured 
into Petri dishes and placed in the incubator. It is advisable to use both 
solid and fluid media. Anaerobic cultures are prepared as usual, using, 
preferably, a combination of a vacuum with alkalinized pyrogallic acid solu- 
tion to insure absence of oxygen. 

If, after incubation for 24 hours, the plates show only a few surface col- 
onies, contamination is to be considered, while if the colonies be deep and 
are observed in several plates, contamination may be ruled out. This must 
not be interpreted to mean that any colonies will, necessarily, develop within 
24 hours, as the plates may have to be kept for 5 to 10 days* before a negative 
result may be certain. However, the contaminating organisms usually show 
up within 24 hours. The fluid media will become cloudy if any organisms are 
present. From the plate colonies and the fluid media sub-cultures are then 
made upon different media and every expedient possible used to isolate and 
identify the offending organism. Usually the infecting organism may be 
obtained in pure culture as a mixed infection in the blood is very uncommon. ^ 



Organisms Found in the Blood. 
I. Bacillus Typhosus. 

Instead of transferring the blood, withdrawn from the vein, to bouillon 
or melted agar, one may add it directly to sterilized ox-bile in the propor- 
tion of I or 2 c.c. of blood to 5 of the bile. The blood may, if desired, be 
obtained in this case from a puncture of the ear (the puncture being prefer- 
ably made through a coating of collodion and the blood allowed to drop 
directly into the tube of bile). The mixture of bile and blood is allowed to 
incubate at 37°C.for 12 to 15 hours, when transfers of a fewloopsful are made 
to the Drigalski-Conradi or modified Endo media discussed under Feces. 
The characteristic colonies are then sub-cultured and the agglutination tests 
made as described in the next section. 



^ Hiss and Zinsser (Text-book of Bacteriology, p. 126) recommend the addition of a 
small piece of calcium carbonate to the liquid media in order to neutralize any free acid 
formed in case the pneumococcus is suspected. See, also, Gillespie (Jour. Exper. Med., 
1 9 13, XVIII, 584) for comparative viability on fluid and solid media. 

2 Warren and Herrick, Am. Jour. Med. Sc, 1916, CLI, 556: Bloom, Am. Jour. Dis. 
Child., 191 7, XIII, 128; Wurtz and Sappington, Jour. Med. Res., 1918, XXXVIII, 371; 
Richey and Goehring, Ibid., 421; Chamberlin and Minaker, Jour. A. M. A., 1919, LXXII, 
1073- 



THE BLOOD 649 

According to Kolle and Hetsch/ this bacillus of Eberth-GafFky shows 
the following characteristics: it is an actively motile bacillus with numerous 
flagella, stains wdth the ordinary dyes but is Gram-negative, produces no 
indol in bouillon (colon bacillus does), produces no gas in glucose-bouillon 
(colon bacillus does), produces no acid nor any coagulation in litmus-milk 
(colon bacillus produces much acid, while the bacillus faecalis alcaligenes is a 
strong alkali-former), produces no change in neutral red-agar (colon bacillus 
and paratyphoid bacillus B cause fluorescence of the solution and gas forma- 
tion, while the bacillus faecalis alcaligenes and the dysentery bacillus produce 
no effects), produces dew-drop colonies on litmus-lactose-agar with no change 
in color of the media (colon bacillus produces marked red coloration), pro- 
duces acid and coagulation of the casein in litmus-nutrose-glucose solution, 
while no such effects are observed in litmus-nutrose-lactose solution, shows 
characteristic agglutination with pure culture of typhoid bacillus. 

Ordinarily the agglutination tests are all that are necessary after the 
organism has been grown on the Drigalski-Conradi or other media. However, 
it is to be remembered that the freshly isolated organisms show less tendency 
to agglutinate than do the organisms w^hich have been grown for some time 
or have been transferred through several generations. 

The importance of blood cultures ^ in cases of typhoid fever may be esti- 
mated by the experimental evidence that 70 to 80 per cent, of cases are posi- 
tive, oftentimes many days before the agglutination tests are positive. It 
has been further shown that the organisms tend to diminish in the blood 
during the course of the disease until they practically disappear from the 
blood about the end of the third week. 

2. Bacillus Paratyphosus. 

In certain cases, w^hich clinically closely resemble typhoid fever, although 
running a somewhat milder course, organisms may be isolated from the blood, 
which are intermediate between the true bacillus typhosus and the bacillus 
coli communis. The clinical differentiation of these cases is often difficult, 
although the sudden onset of vomiting, diarrhea, chill, and fever of paraty- 
phoid are somewhat different from the more gradual onset and step-like in- 
crease of temperature in typhoid; while the temperature-curve, the somewhat 
different early characteristics of the feces, the early appearance of herpes, 
the usual lack of splenic enlargement, etc., may serve as differential points 
in favor of paratyphoid.^ Aside from this type, we find sporadic or epidemic 
cases in which the symptomatology is that of a severe gastroenteritis, the 

^ Die Experimentelle Bakteriologie. Berlin, 1911; Demolon, Paris Med., 1916, VI, 
354: Vaughan, Jour. Lab. and Clin. Med., 1919, IV, 640. 

2 This work is fast becoming an absolute necessity, as the employment of the preventive 
inoculation for typhoid practically destroys the reliability of the Widal test of the serum of 
those previously inoculated. See Moreschi, Ztschr. f. Immunitatsforch., 1914, XXI, 410; 
Mcintosh and McQueen, Jour. Hyg., 1914, XIII, 409; Saski, Ztschr. f. klin. Med., 1914, 
LXXX, 79; Carnot and Weill-Halle, Presse med., 1915, XXIII, 89; Hohlweg, Miinch. 
med. Wchnschr., 1915, LXII, 538; Lipp, Ibid., 539. 

2 See Hunt, Arch. Int. Med., 1913, XII, 64; Heigel, Wien. klin. Wchnschr., 1915, 
XXVIII, 57; Dreyer, Walker and Gibson, Lancet, 1915, I, 324 and 643; Irons and Jordan, 
Jour. Infect. Dis., 1915, XVII, 234; Robinson, Jour. Med. Research, 1915, XXXII, 399; 
Bernstein and Fish, Jour. A. M. A., 1916, LXVI, 167. 



I 



! ^ 



ii 



650 DIAGNOSTIC METHODS 

so-called "infectious meat-poisoning" usually classed as ptomaine poison- 
ing, which is due to the bacillus para typhosus. These organisms are widely 
spread in the animal kingdom and may be found even as a contamination in 
water supplies. Not infrequently it is found in man, as a pure saprophyte, no 
clinical symptoms traceable to its presence being observed. It may be 
present in the pus of otitis media, orchitis, cholecystitis, osteochondritis, 
arthritis, lymphadenitis, also, as a secondary invader, in scarlet fever, 
measles, pneumonia, meningitis, etc. 

The examination of the blood is made as previously described, transfers 
and sub-cultures being made upon the various media. These paratyphoid 
bacilli are, apparently, of two types, known as "A and "B." Both types 
differ from the bacillus typhosus in that they produce gas in glucose media 
and show different agglutination reactions. Type A behaves in most other 
respects like the typhoid bacillus, except that the former causes fluorescence 
in neutral-red agar. Type B is differentiated from the typhoid bacillus and 
from type A of the paratyphoid bacillus by not coagulating litmus-milk 
but in producing acid (red coloration) and, after 5 to 10 days, in causing a 
conversion of this red color to a deep blue (due to alkali formation). The 
typhoid, colon, paratyphoid A and dysentery bacilli never cause a blue 
coloration of the milk, while the blue color due to the bacillus faecalis 
alcaligenes is produced at once. Jordan^ has shown that there are cultural 
differences between the Paratyphoid A and B strains and those of the 
enteritidis group, these being manifest in the formation or absence of forma- 
i, tion of acid and gas in xylose, arabinose and dulcite, and in the degree of 

' rapidity of alkali formation in litmus milk. The paratyphoid A strains, 

which are derived from human sources, produce alkali in litmus milk much 
more slowly than the others and do not ferment xylose. The paratyphoid 
j B strains ferment xylose, arabinose and dulcite rapidly and produce alkali 

i quickly in litmus milk. The enteritidis strains are indistinguishable from 

I the paratyphoid strains B, but have distinct agglutinative reactions. The 

t ' peculiarities of the agglutination reaction with this organismwill be discussed 

in the next section. 

\ 3. Bacillus Coil Communis. 

This organism is, probably, not a unitary organism, as many types of 
the species exist. The colon bacillus is found in all warm-blooded animals 
and in many cold-blooded ones as a normal habitant of the intestinal canal. 
These bacilli show a marked antagonism for the putrefactive bacteria, so that 
physiologically, they should be regarded as conservative organisms. Owing 
, to the wide occurrence of this organism, it is frequently found as a contamination 

of food and water supplies unless special precautions are taken to exclude it. 
Although ordinarily a harmless type, yet, from the pathologic stand- 
point, the colon bacillus is generally regarded as a pathogenic organism under 

^ Jour. Infect. Dis., 1917, XX, 457. See, also, Krumwiede, Pratt, and Kohn, Jour. 
Med. Res., 1916, XXXIV, 355; Ibid., XXXV, 55 and 357; Weiss and Rice, Ibid., 403; 
Labbe and Canat, Ann. de Med., 191 7, IV, i; Jordan and Irons, Jour. Infect. Dis., 1918, 
XXIII, 537; Krumwiede, Kohn, and Valentine, Jour. Med. Res., 1918, XXXVIII, 89; 
Ibid., 1919, XXXIX, 449. 



THE BLOOD 65 I 

certain conditions. In this connection we must distinguish an endogenous 
from the exogenous colon infections. The first are found in cases of wound 
infection and, more especially, in infections of the lower urinary tract into 
which the bacilh have gained entrance through the urethra; while the latter 
are observed under the following conditions. So long as the natural protective 
and bactericidal agents of the body are intact and so long as the intestinal 
epithelium is unimpaired, the colon bacilli do not pass into the organs or 
serous cavities of the body. However, in the course of any disease, such as 
typhoid, cholera and severe non-specific enteritis, in which the epithelium is 
destroyed, the colon bacillus may pass from the intestinal tract, either by 
direct contiguity of tissue or through the blood current, and assume a 
pathogenic role as a secondary invader. Moreover, should the patient be 
suffering with a chronic wasting disease which lowers the general resistance to 
infection, the continuity of the epithelium may still be preserved and the 
bacillus become pathogenic in many organs. Thus we find infections due to 
the bacillus coli communis in the kidney, urinary bladder, gall-bladder, peri- 
toneal cavity, ovary, uterus, and other organs. Pus pockets, which may have 
caused little clinical disturbance, may spread, become chronic and induce a 
septicemia, of which the etiologic factor may be determined only by resort to 
blood culture. 

The biologic properties distinguishing the colon bacillus from the typhoid 
and paratyphoid bacilli are as follows: The colon bacillus is very little, if 
at all, motile; it produces gas in glucose media and indol in bouillon or pep- 
tone media; it coagulates milk and produces acid with red coloration in litmus 
milk; its colonies on litmus-lactose agar are deep-red in color (the color diffus- 
ing into the surrounding media), while those of typhoid and paratyphoid 
are colorless; in neutral-red agar it causes fluorescence and marked gas pro- 
duction, while the typhoid bacillus produces no change in the media; it does 
not show characteristic agglutination reactions, while the typhoid bacillus does. 

4. Pneumococcus. 

This organism may be recovered from the blood in practically all cases of 
lobar pneumonia due to infection with this type, although it must be remem- 
bered that other organisms may be the etiologic factor in some cases. At 
times the pneumococcus may be obtained as early as 1 2 hours after the initial 
chill, while in other cases it may appear as late as 48 hours after the crisis. 
Rosenow obtained positive results in 160 out of 175 cases, Prochaska 48 out of 
50, while Cole and Lenhartz report only 30 per cent, of positive findings. ^ The 
finding of the pneumococcus in the blood does not, necessarily, indicate an un- 
favorable prognosis, although a large percentage, 40 to 50, of the cases in which 
it is found result fatally. ^ The earlier the organisms are found the more 
grave the prognosis. The number of organisms found by culture is not as 
large as in other types of blood infection, so that Fraenkel is lead to be- 
lieve that the prognosis is bad when a large number of colonies develop from 
I c.c. of blood. The infection may spread through the lymphatic channels 

1 See Lyall, Jour. Am. Med. Assn., 191 2, LVIII, 1841. 

2 See Hastings and Boehm, Jour. Exper. Med., 1913, XVII, 239. 



652 



DIAGNOSTIC METHODS 



to the blood, so that we may find these organisms in the bile, urine, milk and 
other body secretions. Moreover, we may have many infections in other 
organs as a complication of the original pneumonia. 

Oftentimes, we find the pneumococcus as the cause of primary patho- 
logical conditions in many organs, when there has been no antecedent pneu- 
monia. Thus we may have a pneumococcic otitis media, bronchitis, pleuritis, 
peritonitis, meningitis, endocarditis, pericarditis, orchitis, tonsillitis, cystitis, 
salpingitis, enteritis, conjunctivitis and keratitis. The causative factor may be 
discovered only by examinations of the various excretions related to the special 
condition. While the blood culture may be of little clinical value in the diag- 
nosis of lobar pneumonia as the clinical findings are usually so clear-cut, yet it 
may be the only means at our disposal in solving the mystery of an obscure 
infection in other parts of the body, as for instance in a malignant endocarditis.^ 

The blood is taken as previously outlined and is transferred to agar, 
blood-agar, plain, serum or ascitic bouillon, or litmus-milk. The organisms 
which are especially confusing from the standpoint of differentiation are the 
various types of streptococci. On blood-agar plates the pneumococcus shows 
an especially characteristic greenish hemolytic colony. The streptococcus 
viridans produces the same green zone, while the streptococcus pyogenes 
shows no green color but a large colorless hemolytic zone. Blood bouillon 
shows no hemolysis with either the pneumococcus or streptococcus viridans, 
while a marked burgundy-red color appears in the presence of streptococcus 
pyogenes. The differentiation between the pneumococcus and streptococcus 
viridans is made by the use of bile acids. Bouillon cultures of pneumococcus 
are cleared while those of the viridans are unaffected. Further, the pneu- 
mococcus ferments inulin solutions, while most strains of streptococci do not 
produce this result. The detection of the capsule and the agglutination re- 
actions clear up the diagnosis. 
Rosenow's Capsule Stain. 

This method^ is, in the writer's opinion, the best at our command for 
this purpose. The technic is as follows: Make a thin smear on a perfectly 
clean slide or cover-glass. If the material, such as sputum, is too thick, add 
enough distilled water so that it can be spread evenly by means of a piece of 
fine tissue or cigarette paper. In cases of cultures (blood-agar, serum, glu- 
cose or Loeffler's blood-serum being preferable), remove a small amount of the 
growth from the surface of the medium and at once mix thoroughly with a 
loopful of serum on the slide, or, better still, make a rather dense suspension 
in a few drops of distilled water and then mix an equal quantity of this 
suspension with serum, and spread by means of tissue paper. As the smear 
becomes nearly dry, cover for 10 to 20 seconds with 5 to 10 per cent, aqueous 
solution of tannic acid; wash in water and blot; stain with carbol (saturated 
alcoholic solution gentian violet, i part, 5 per cent, aqueous carbolic acid, 
4 parts) or anilin gentian violet half a minute to a minute, heat over flame 

^ See Rosenow, Illinois Med. Jour., 191 2, XXI, 425; Jour. Infect. Dis., 191 2, XI, 94; 
also, Cole (Jour. Am. Med. Assn., 191 2, LIX, 693), for a discussion of immunity in pneu- 
monia; Cole, Arch. Int. Med., 1914, XIV, 56; Day, 111. Med. Jour., 1916, XXIX, 354. 

2 Jour. Infect. Dis., 1911, IX, i. 



THE BLOOD 653 

but do not boil; wash in water again; Gram's iodin solution for }/^ to i minute; 
decolorize in alcohol (95 per cent.) ; stain for 2 to 10 seconds, depending on the 
thickness of the smears, with saturated alcoholic (60 per cent.) solution of 
Griibler's eosin; wash in water and blot finally, clear in xylol and mount in 
balsam or examine directly. If the organism, like the Bacillus mucosus, 
is Gram-negative, the bacillus may be stained with Loefller's or aqueous 
methylene blue. 

The pneumococci are stained deeply brownish-black, sharply differenti- 
ated from the capsule, which is stained pink. Beautiful results are also ob- 
tained with the streptococcus mucosus. In the thickest part of the smear 
the space occupied by the capsule may be perfectly clear; elsewhere in the 
smear, if properly made, where the conditions are suitable for absorption 
of eosin, the capsule is stained deeply pink; not rarely a clear refraction 
zone (often mistaken for the capsule in former methods) may be seen periph- 
erally to a distinctly stained, often large capsule. In case of sputum in 
which the cocci are embedded in a more or less tenacious mucus, the cap- 
sules, at times, are not rendered stainable by the above method. In that 
case it is well to fix and stain simultaneously with the 2 per cent, aqueous 
tannic acid, 4 parts, and saturated solution of gentian violet, i part. This 
modification often gives beautiful results. The cocci, however, decolorize 
easily and the tannic acid-gentian-violet may be followed by carbol-gentian- 
violet and then the usual procedure. Ordinary carbol-fuchsin, diluted five 
to ten times, and aqueous eosin (50 per cent, saturated solution) may also be 
used to stain the capsule, although the saturated alcoholic (60 per cent.) eosin 
has given the best results. Decolorization after the modified Gram pro- 
cedure of tannic acid-fixed smears is more rapid than in the case of heat- 
fixed smears, which fact should be borne in mind. 

By the use of this method it has been determined that the capsule of the 
pneumococcus and allied organisms is not difficult to preserve or readily 
soluble in water, as is generally believed. To stain the capsule is a problem of 
rendering it stainable rather than one of preservation. The reactions which 
accomplish this appear to be colloidal reactions. 

5. Streptococci. 

In general infections with any of the varieties of the streptococcus, blood 
cultures reveal the offending organism in a large percentage of cases, de- 
pending, of course, upon the location and severity of the process. There 
are many varieties of streptococci, of which some are saprophytic and some 
pathogenic. Pathologically, streptococci are found in the blood in many con- 
ditions. They may obtain entrance to the blood by rupture of a streptococcic 
abscess into a vessel. Infected thrombi, as met with in puerperal sepsis, may 
form the source of the invasion. Long-standing infections, such as empye- 
mas, may so lower the resistance of the patient that a septicemia may result. 
An especial point of invasion is through wounds, the infection here often 
being extremely dangerous. A further type is the cryptogenetic septico-pye- 
mia in which the source of infection can not be traced. 



ii>l 



654 



DIAGNOSTIC METHODS 



The organism may be obtained from the blood in practically all cases 
of mycotic endocarditis due to this streptococcus. In puerperal sepsis the 
results are not so frequent, only about one-fourth of the cases being positive. 
The cryptogenetic types associated with metastatic lesions in the joints and 
organs show a much larger percentage of positive findings. In scarlet fever 
we find, according to Hektoen, that streptococci may occasionally be found 
in the blood in cases which run a short, mild, and uncomplicated course; they 
occur with relatively greater frequency in the more severe and protracted 
cases in which there may develop local complications and signs of general 
infection, although they may not be demonstrable in fatal cases. The strep- 
tococci may also be found in diphtheria, measles and small-pox especially in 
the fatal cases, in which they act as secondary invaders. It is to be em- 
phasized that the prognosis is not, necessarily, bad when streptococci are 
found in the blood, although there is a large mortality. 

The morphology of these organisms is variable and the varieties numer- 
ous. ^ The pathogenic types, isolated directly from pathologic lesions, appear 
to consist of rather long chains of more than 8 pairs of cocci when grown in 
bouillon, while the non-pathogenic types form shorter chains. Involution 
forms are common in specimens from culture. Some of them are encap- 
sulated, two organisms being enclosed. This may lead to confusion with 
the pneumococcus but the differential points given under the pneumococ- 
cus will serve to distinguish them. Usually the microscopic examination of 
the stained specimen will be all that is necessary, but occasionally careful 
work is needed to identify them. In doubtful cases, )^ to i c.c. of the 
blood withdrawn by venous puncture may be injected intraperitoneally 
into a mouse, when a general streptococcic septicemia will ensue. 

Many other organisms have been and may be isolated from the blood. 
Thus the staphylococci are not infrequently the cause of a bacteriemia arising 
from an endocarditis, osteomyelitis, or furunculosis. The gonococcus is 
often found in cases associated wtih gonorrheal endocarditis, arthritis, paro- 
titis, myositis, etc. The meningococcus, the influenza bacillus, bacillus 
mucosus, bacillus of anthrax and of glanders have been obtained from the 
blood in suitable cases. ^ The tubercle bacillus has been repeatedly found in 
cases of both acute and chronic tuberculosis. However, much recent work 
since the publication of Rosenberger, who attempted to prove that tubercle 

1 See Winslow, Jour. Infect. Dis., 191 2, X, 285; Jungmann, Deutsch. Arch. f. klin. Med., 
1912, CVI, 283; and Bergey, Jour. Med. Research, 1912, XXVII, 67. See p. 41 for a 
discussion of streptococcic sore-throat. Also, Thalhimer and Rothschild, Jour. Exper. 
Med., 1914, XIX, 429 and 444; Simons, Quart. Jour. Med., 1914, VII, 291; Rosenow, Jour. 
Am. Med. Assn., 1914, LXIII, 903; Oille, Graham and Detweiler, Ibid , 1915, LXV, 11 59; 
Rosenow, Jour. A. M. A., 1915, LXV, i687;Lintz, Jour. Lab. and Clin. Med., 1918, III, 

509- 

2Tunnicliff (Jour. A. M. A., 1917, LXVIII, 1028) has isolated from the blood m pre- 
eruptive and eruptive stages of measles, by using anaerobic cultures, a small, round, 
sometimes flattened diplococcus, which may appear in short chains, sometimes as clumps 
of cocci of varying sizes. Reed (Jour. A. M. A., 1914, LXIV, 1047; Ibid., 1916, LXVI, 
336) announced the finding in the blood of a coccus, the epilepticoccus which he believed 
to be the cause of epilepsy. Later he (Ibid., 1607) described a large spore-bearing organ- 
ism styled bacillus epilepticus as the etiologic factor. Wherry and Oliver, Ibid., 1916, 
LXVII, 1087) identify this organism as the bacillus subtilis. See, also Caro and Thom, 
Ibid., 1088; Terhune, Ibid., 1155. 



THE BLOOD 655 

bacilli were present in the circulating blood of incipient tuberculosis and that 
a primary bacteriemia was always present in these cases, has absolutely re- 
futed this finding and has emphasized the importance of eliminating the 
possibility of the presence of acid-fast bacilli in the reagents and, even, in 
the distilled water used.^ 

It is probably true that the organism at the bottom of every infectious 
disease of known origin may be obtained from the blood at some stage of the 
infection. It is important, therefore, that the clinical examination include 
blood cultures in any case of obscure origin, as in this way treatment and 
prognosis will be markedly influenced. Churchill and Clark^ have recently 
called attention to the importance of this work in children. 

VII. Serum Pathology 

This section of hematology is very closely associated with pathology and 
biochemistry and can, therefore, be taken up only in brief outline.^ As a 
matter of fact, changes in the number of the cells as well as in the percentage 
of hemoglobin must be dependent, to a certain extent, upon the more obscure 
changes which are taking place in the plasma in various diseases. Our ability 
to fathom the secrets of the many physical and chemical changes of the plasma 
has been so slight that we have hitherto neglected to take into consideration 
anything but the changes in the cells, which can be so easily studied by the 
various methods previously outlined. Our knowledge of the various types 
of immunity to infection and of the many factors concerned therein has in- 
creased to such an extent in recent years that a complete discussion is out of 
place here. By the elaboration of the side-chain theory of Ehrlich and of the 
opsonic theory of Wright, we have come somewhat nearer to a proper realiza- 
tion of the importance of the serum in all infections as well as in many diseases 
in which great metabolic disturbance is evident. For that matter one can 
hardly imagine a condition in which the blood plasma may not show some 
characteristic change, inasmuch as the nutrition of the entire body can come 
through the blood. When one considers the close correlation of the various 
organs he may see at once that pathological changes in any of the viscera may 
result in an abnormal blood, which may show no variations at present capable 
of detection. For these reasons one hails with delight any advance in serum 
pathology and could but wish that his knowledge might more rapidly increase. 

^ See Berry, Jour. Infect. Dis., 1914, XIV, 162; also, Rautenberg, Berl. klin. Wchnschr., 
1914, LI, 348 and 492; Baetge, Deutsch. med. Wchnschr., 1914, XL, 591; Faber, Jour. Am. 
Med. Assn., 1914, LXIII, 1656; Jousset, Bull. I'Acad. de Med., 1915, LXXIII, 203; Luc- 
ciarini, Rif. Med., 1915, XXX, 253 and 281; Austrian and Hamman, Bull. Johns Hopkins 
Hosp., 1915, XXVI, 293; Kessel, Am. Jour. Med. Sc, 1915, CL, 377; Honeij, Jour. Infect. 
Dis., 1915, XVII, 376; Wilson, Jour. Infect. Dis., 1916, XIX, 260; Hall and Harvey, Jour. 
Med. Res., 1917, XXXV, 265. 

2 Am. Jour. Dis. Child., 1911, I, 193. 

^ See Kraus and Levaditi, Handbuch der Technik und Methodik der Immunitats- 
forschung, Jena, 1909-1911; Citron, Die Methoden der Immunodiagnostik und Immuno- 
therapie, Leipzig, 191 2; Dieudonne, Immunitat, Schutzimpfung und Serumtherapie, Leip- 
zig, 1913; Vaughan, Protein Split Products in Relation to Immunity and Disease, Phila- 
delphia, 1913; Kolmer, Infection, Immunity and Specific Therapy, Philadelphia, 1915. 



656 



DIAGNOSTIC METHODS 



Ehrlich's Side-chain Theory. ^ 

The early work of Ehrlich, published in 1885, advanced a theory to ac- 
count for various phases of immunity, especially of the action of the blood in 
producing antitoxins against various poisons elaborated by infectious agents. 
It is to be said that no such formation of antitoxin against the ordinary medic- 
inal poisons has been found. 

According to this theory, the protoplasm of the cell consists of a central 
group of molecules (Leistungskern), in which the inherent vital characteristics 
of the cell are located and whose integrity is necessary for normal cell life. 
At different portions of the cell certain other molecular groups are attached 
exactly as side-chain groups are attached to the benzene nucleus of organic 
chemistry. These groups or, as EhrHch styles them, side-chains are capable 
of uniting with various material which is brought into intimate relationship 
with the cell structure. Such materials are foods, toxins, and other injurious 



Toxin united with cell. 



Receptors. 




^^- -— 'pQxophore. 

1/ \|- Haptophore. 

Toxin molecule. 



Body cell 
Fig. 150. — Illustrating the mechanism of the toxin-cell union by the intermediation 
of receptors. {Da Costa.) 

agents. In order that foods may be taken up by the cell it must possess cer- 
tain groups which will enable it to combine with the groups in the side-chain 
of the cell. It must be, in other words, homologous or, as Ehrlich states, 
must bear the same relationship to the side-chain which Fischer has applied 
in his assumption of the "key in the lock" hypothesis regarding ferment 
action upon the various types of hexoses. In the nomenclature of Ehrlich 
the side-chains are styled receptors and the group of the food or of the toxin 
which combines with these receptors is known as the haptophore group. 
These receptors, as well as haptophores, possess specific affinity, uniting with 
one another only when homologous. 

It has been found that a toxin molecule has certain injurious effects upon 
the cell; it is, therefore, necessary to ascribe this action to other than the 
two groups above mentioned, as the union of a haptophore with a receptor 

^ In this connection see Sachs, Die Haemolysine, Wiesbaden, 1902; Kraus andLevaditi, 
Handb. d. Tech. u. Meth. d. Immunitatsforschung, Jena, 1907-1911; Adami. Principles of 
Pathology, New York, 1910; Dieudonne, Immunitat, Schutzimpfung und Serumtherapie, 
Leipzig, 1913; Kolle and Wassermann, Handbuch der pathog. Mikroorg., Jena, 191 2; 
Koile and Hetsch, Die Experimentelle Bakteriologie, Berlin, 191 1. 



THE BLOOD 



657 



would form an inert substance. Ehrlich, therefore, assumes the presence in 
the toxin^ molecule of a second group which he styles the toxophore group, 
which exerts the untoward effect upon the cell. The toxophore group in it- 
self can unite with the cell only through the medium of its haptophore group. 
As the cell becomes irritated by the presence of the toxophore group, it 
endeavors to overcome this by a new formation of receptors. According to 
the strength of the irritation, many more receptors will be found that can 
combine with the toxin material present, so that many of the extra receptors 
pass out into the circulation in the form of free receptors. This is a graphic 
explanation of the fact that the cell when irritated by toxic material elabo- 
rates substances from its own protoplasm, which have a neutralizing effect 
upon the toxic substance. These free receptors (haptines) form the anti- 
toxins. They combine only with homologous toxin material and are, there- 
fore, specific. Welch believes that these antitoxins have a second function 



Toxins united 

with cell. 



Free recep- 
tors; haptin; 




Toxins united with 
antitoxin. 



Body cell. 
Fig. 151. — Illustrating the elaboration and action of antitoxin. {Da Costa.) 

beside that of neutralization of toxin, namely, an irritating one upon the bac- 
terial invaders so that these organisms are forced to elaborate similar sub- 
stances for their own protection. Toxins which have been deprived of their 
toxophore group are known as toxoids and can combine with the receptors 
of the cell, exerting no untoward effect upon the cell. These toxoids may also 
unite with antitoxin through the medium of their haptophore group. Occa- 
sionally toxins are incompletely combined with the antitoxins; that is, the 
antitoxic material is not sufficient in amount completely to neutralize the 
toxin, so that such toxins may still combine with the cells and exert a mod- 
ified poisonous effect. Such attenuated toxins are known as toxones. 

It has been found that the injection into animals of bacteria, various 
body cells, and certain secretions of some animals, as for instance, snake 
venom, gives rise to the development of specific antibodies in the blood serum 
of the animal so treated, the substances injected being named antigens.^ 

^See Coca, Jour. Infect. Dis., 1915, XVII, 351. 

2 See Pick (in Kolle and Wassermann's Handb. d. pathogen. Mikroorg., 191 2, I, 685) 
who shows that true antigens are always albumin-containing colloids. Also, Elliott, Jour. 
Infect. Dis., 1914, XV, 501; Salus, Biochem. Ztschr., 1914, LXVII, 357; Porter, Biochem. 
Jour., 1915, IX, i; Banzhaf, Sugiura and Falk, Jour. Immunol., 1916, II, 125. 
4 



658 



DIAGNOSTIC METHODS 



r 

I 



Such blood or serum will be found to have a lytic (destructive) action upon 
cells similar to those injected. Such sera are specific, that is, they act only 
upon the kind of cell used in the injection. The term hemolysis has been 
introduced to express the destructive action upon the erythrocytes shown by 
the dissolving out of the hemoglobin from the red cell. The stroma or dis- 
coplasm of the red cell is a membrane which shows peculiar relations to diffu- 
sion of various materials into the cell and to the passage of hemoglobin and 
other cellular material from the cell. Its chief function seems to be to pre- 
vent, as far as possible, any loss in hemoglobin. If this membrane becomes 
permeable, then we must assume the action of some toxic material. The 
term hemolysis has reference merely to the abnormal loss of hemoglobin and 
not to any disturbance beyond increased permeability of the stroma. The 
stroma of these cells remains behind and may be seen in the centrifuged speci- 
men as the so-called shadows. Hemolysis must, therefore, be considered 
as a sign of protoplasmic death. Substances (hemolysins) bringing about 



Union of complement, ambo- 
ceptor, and cell. 



Receptors 




— - Zymophore. 

.Haptophore. 

Complement 



u 



— Complementophile. 

Cytophile. 

Amboceptor. 



Erythrocyte. 
Fig. 152. — Illustrating the mechanism of hemolysis. {Da Costa.) 

such change belong, necessarily, in the class of blood poisons. Such hemoly- 
sins are increased or lowered temperature, various inorganic compounds, such 
as distilled water, ammonium salts, and organic compounds, such as urea, 
bile acids, ether, alcohol, chloroform, solanin, saponin, and digitalin. The 
saponms are among our strongest hemolytic substances, acting in dilutions of 
I to 100,000. Besides these we have various secretions, such as those of 
the cobra, spider, and the bees which are active hemolytic agents. 

It has been observed by various workers that hemolysis is prevented when 
the serum is heated to 60°. This points to the fact that some substance is 
destroyed which is of great importance in this process. In addition, it has 
further been found that the renewing of the activity of the old heated serum 
by adduig a supply of fresh isologous serum will restore the hemolytic activity. 
It is evident, therefore, that there are two factors which must be taken into 
consideration, one is a thermostable (heat-resisting) substance, while the 
other is thermolabile (destroyed by heat). To the first of these Ehrlich has 



THE BLOOD 659 

given the name amboceptor and to the second the name complement. The 
amboceptor has been shown to have two haptophore groups, with one of 
which its unites to the receptor of the cell and with the other to the comple- 
ment. The haptophore group which unites with the cell is known as the 
cytophile, while the one uniting with the complement is termed the comple- 
mentophile. For hemolysis, therefore, we must have the cell receptor, the 
amboceptor, and the complement. The complement has also been shown to 
have two groups analogous to those of the toxin molecule. The first is the 
haptophore group, while the second is the zymophore group, through which 
the destructive action upon the cell is manifest. 



Receptors. 





Erythrocyte. 

Fig. 153. — Illustrating the Mechanism of Antihemolysis. {Da Costa.) 
A, Interference of anticomplement with complement-amboceptor union. B, inter- 
ference of antiamboceptor with amboceptor-cell union. C, antiamboceptor-amboceptor 
union. D, anti-complement-complement union. 

The amboceptor is formed within the body as the result of cellular 
hyperactivity aroused by the irritant action of the toxic material. The com- 
plement is probably derived, for the most part, from the leucocytes, and acts 
Very much as an enzyme. It can exert its toxic action only when united 
with the cell by means of the amboceptor, so that free complement has no 
injurious effect. 

It has been frequently observed that the red cells are more resistant than 
normally, while in many cases they appear less resistant to hemolysis. This 
is explained by the side-chain theory very much as it explains the formation of 
antitoxin. These antihemolysins are formed within the blood plasma after 
inoculation with hemolytic material. The hyeractivity of the cell causes 
it to throw off two types of such bodies, namely, anticomplement and anti- 
amhoceptors. The former combines with the haptophore group of the comple- 
ment and the latter with the cytophilic group of the amboceptor, each of these 
combinations making it impossible for the necessary union of cell, amboceptor, 
and complement to occur. 



66o DIAGNOSTIC METHODS 

It has been found that frequently the serum of an animal, which has been 
injected with certain bacteria or with certain body cells, shows the peculiar 
property of agglutinating or clumping such bacteria or cells when these latter 
are added to it. This condition is known as agglutination and the agents 
bringing it about are st^ded agglutinins. These substances are developed in 
the blood of the animal during the process of adaptation toward the presence 
of such foreign material. Agglutinins from the standpoint of the side-chain 
theory are free receptors, having a haptophore group which unites with the 
receptor of the cell or of the bacterium, and cause agglutination through the 
presence of a second group known as the zymophore or agglutiniphore group. 
Agglutination, therefore, does not require the presence of a complement. 

In some cases, intraperitoneal injection of body fluids or of solutions of 
certain proteins into animals brings about a condition which enables the 
blood serum of such animals to cause a precipitation of the protein to which 
the animal has been adapted. This fact has been taken advantage of in 
formulating a medicolegal method for the detection of blood of different ani- 
mals and will be taken up in a later section. The precipitins consits of free 
receptors combining, by means of their haptophore group, with the receptor 
of the cell and exerting their precipitating effect through the medium of their 
zymophore or precipitinophore group. 

Phagocytosis. 

According to the theory of Metchnikoff, the leucocytes are capable of 
incorporating into their substance materials which are foreign to the blood 
in which they circulate. This process is known as phagocytosis and is one 
of the greatest protective measures which the system has for its fight against 
bacterial invasion. When the blood becomes laden with bacteria, as in the 
various infectious diseases, we find a leucocytosis in practically all cases, the 
exceptions having been previously noted. This is the natural sequence if 
the system is to rid itself of these invaders. The leucocytes are drawn by 
chemical attraction (chemotaxis) toward the bacteria and attempt to swallow 
them by throwing pseudopodia about them and drawing them into the proto- 
plasm. This is successful in many cases, while in others it is not, so that the 
question of ascendency of the leucocyte or of the bacterium will depend upon 
the degree of phagocytosis. Strangely enough much variation is shown in the 
susceptibility of different organisms to phagocytosis. Thus the pneumococ- 
cus at times is very difficultly amenable to phagocytosis, while at others it is 
easily acted upon. The recent work of Rosenow^ has thrown much light 
upon the mechanism of this action. 

Opsonins. 

Realizing that there was a more definite basis for phagocytosis than was 
embraced in the older conceptions, Wright^ introduced the idea of opsonins 
to designate the presence in the blood serum of substances which render the 

1 Jour. Infect. Dis., 1906, III, 683; also, Kite and Wherrv, Ibid., 191 5, XVI, 109; Tunni- 
cliff, Ibid., 1916, XIX, 97; Douglas, Proc. Royal Soc. London, 1916, LXXXIX. 335; Wolff, 
Xederl. Tijdschr. v. Geneesk., 1916, II, 1789. 

- Proc. Royal Soc, 1903, LXII, 357. 



I 



THE BLOOD 66 1 

various bacteria subject to phagocytosis. The normal blood serum contains 
such opsonic material for the various bacteria with which it may be infected, 
but this varies greatly toward the different organisms. Thus we may find 
individuals showing much more opsonin (a higher opsonic index) toward one 
organism than toward another. This explains, in a way, the well-known fact 
that different people are variably susceptible to the same disease, while the 
same individual may be strongly resistant toward infection with one organism, 
but easily a victim of another infection. 

Regarding their chemical nature very little is known. There seems to be 
a certain amount of evidence which points to the fact that these opsonins 
belong to the class of globulins, while Simon and Lamar^ have shown that 
they are apparently associated with the euglobulin fraction. Quite as little 
is known of the structure of the opsonins, so that it is at present doubtful 
in what position to place them in the side-chain theory. According to Hek- 
toen,2 they may contain a haptophoric group which unites with the bacterial 
or other receptors and also an opsonipherous group which brings about 
changes in the cell, making it capable of phagocytosis. According to Savt- 
chenko and Dean, the opsonins should be regarded as amboceptors, while 
Greig-Smith looks upon the process of opsonification as the first stage of ag- 
glutination. All of these theories must wait for future confirmation. The 
opsonins are thermolabile and are usually destroyed by heating for lo minutes 
to 6o°C. They occur in all classes of vertebrates and show here a peculiar 
characteristic, namely, that the serum of different animals is capable of 
activating various organisms for phagocytosis by leucocytes of animals of 
different species. This would bring the opsonins into the same field as the 
agglutinins, precipitins, and hemolysins. 

From a clinical standpoint, opsonins are frequently found to be diminished 
in certain bacterial infections. It is, therefore, conceivable that the resist- 
ance of the patient or, in other words, his phagocytic power might be increased 
by the addition of substances which could act as opsonic material. Such 
substances are the bacterial vaccines of Wright, suspensions in physiological 
salt solution of dead cultures of the organism to which the patient shows a 
diminished power of phagocytosis. The relation of the phagocytic power of 
the patient, as evidenced by the number of organisms which a definite number 
of leucocytes takes up under the opsonifying influence of this serum, as com- 
pared with the same condition in the case of the serum of a normal individual, 
is known as the opsonic index toward the organism investigated. The 
number of bacteria taken up by the leucocytes of the normal individual is 
taken as one. For preparation of these vaccines see last chapter. 

According to Wright, the injection of a dose of vaccine is followed by a 

decrease in the opsonic index (the negative phase), which is of variable degree 

and duration, depending upon the dose. This negative phase is followed by 

an increase in the opsonic power of the leucocytes (the positive phase) , which 

is associated with improvement in the condition of the patient. The various 

1 Bull. Johns Hopkins Hosp., 1906, XVII, 27. 

^Jour. Am. Med. Assn., 1906, XLVI, 1407; Ibid., 1907, XL VIII, 1739; Illinois Med. 
Jour., XIII, 9; also, Zinsser and Gary, Jour. Exper. Med., 1914, XIX, 345. 



662 DIAGNOSTIC METHODS 

doses of the vaccine should be so administered that it is never given during a 
negative phase. While a low opsonic index is the rule in chronic cases, high 
indices may be observed with active systemic manifestations of acute cases. 
As a rule, it is more generally beneficial to use a vaccine prepared from the 
discharges of the patient than it is to use an already prepared vaccine of the 
same organism. The reason for this is that so much difference exists in the 
various strains of the same organism that little or no result may follow the 
administration of a stock vaccine. It is not always easy to gauge the dosage 
of a vaccine in any particular case, as the idiosyncracy of the patient plays 
such an important role. It is better practice, therefore, to start with a small 
dose and obtain no reaction that it is to give an overdose which will prove very 
harmful. Failure to observe this precaution is, in the writer's opinion, the 
reason that tuberculin so soon fell into disrepute and is now being restored to 
such great favor. 

It has been found by various workers that the administration of a proper 
dose of a vaccine is followed by certain local symptoms, such as swelling, red- 
ness, and tenderness at the point of injection, while certain systemic symp- 
toms, such as rise in temperature, general malaise, and pains in the joints are, 
practically always evident. It has, therefore, become the practice of many 
of our workers, to gauge the results of vaccine treatment more by the reaction 
than by the determination of the opsonic indices of their patients. 

Allergic Reactions. 

The vaccines discussed above are used almost entirely for therapeutic pur- 
poses. While it is highly probable that any of the vaccines might give char- 
acteristic reactions in cases infected with the same organism, yet they have 
not been put to extensive diagnostic use. However, certain suspensions of 
organisms are, at present, widely employed in this way. 

It is evident, from the preceding discussion, that the serum of a patient 
contains bodies acting as neutralizing agents against the toxins of the infect- 
ing bacteria. This property varies with the individual and with the severity 
(dosage), of the infection. Patients, who have acquired immunity against 
certain infectious diseases, either by passing through an attack of the in- 
fection or by use of protective vaccination (small-pox or typhoid fever), 
or who are in the process of acquiring immunity, that is, are at the time pass- 
ing through an active infection, show a certain changed reactivity to in- 
fection with the same organism. In other words such patients are not 
insensible to reinfection, but show a changed reactivity, as von Pirquet^ has 
shown, especially as regards the time, quality and quantity of the reactions 
observed. This is the condition known as Allergy and is shown only by pa- 
tients immune or becoming immune (infected) to the suspected organism. ^ 

Arch. Int. Med., 191 1, VII, 259. 
"^ See. Gurd, Jour. Med. Research, 1914, XXXI, 205; Meigs, Jour. Infect. Dis., 1914, 
XV, 541; Zinsser, Arch. Int. Med., 1915, XVI, 223. Weil, Jour. Exper. Med., 1916, XXIII, 
11; Blackfan, Am. Jour. Dis. Child., 1916, XI, 441; Kolmer and Moshage, Ibid., XII, 
316; Stokes, Jour. Infect. Dis., 1916, XVIII, 402 and 415; Steinfield and Kolmer, Ibid., 
1917, XX, 344; Solmann and Pitcher, Jour. Pharm. and Exp. Therap., 1917, IX, 309; 
Kolmer, Immerman, Matsunami and Montgomery, Jour. Lab. and Clin. Med., 191 7, II, 
401. 



THE BLOOD 663 

Normal individuals do not show the reaction so quickly nor so intensely 
as do the infected or immune subjects, providing the dosage is properly 
regulated, as the former have sufficient antibodies to overcome the effects of 
the amount of toxin, which will produce local or general reactions in the in- 
fected patient.^ These allergic phenomena are due to enzymatic preteolysis 
occasioned by the introduction of the foreign protein into the system and are, 
of course, closely related to the sensitization known as anaphylaxis. Many 
such reactions to food proteins are observed in certain skin diseases.^ 

Tuberculin Reactions. 
Method of Koch. 

This is the earliest method for the diagnostic use of tuberculin. The 
normal temperature variations of the patient are first determined for 24 hours 
previous to the injection. The preparation used is Koch's old tuberculin, 
which is a water and glycerin extract of the cultures of the tubercle bacillus, of 
such a strength that i c.c. is equivalent to i gram of tuberculin. 

The injection maybe made into the deeper tissues either in the intrascapu- 
lar or gluteal region by means of a properly sterilized hypodermic syringe. 
The amount of tuberculin to be injected depends on the age and condition of 
the patient. As a rule 3^^ to i mg. is given to a robust adult, while a weak 
patient or a child receives Ko to %o i^g- These strengths are easily made by 
diluting the original solution with the proper amount of J^ per cent, carbolic 
acid solution. The temperature variations should then be followed, observa- 
tions being made every two hours. A rise of H°C. (%o°F.) within a few hours 
or even as late as 36 hours, is taken as a positive result, although some ob- 
servers require a more marked rise before admitting a positive finding. Few 
other symptoms are noticed but, if the dose has been large, marked malaise, 
chill, high fever, and local reactions may obtain. If no rise in temperature is 
observed after the first injection, a second or a third one may be given, the 
dose being doubled each time but exceeding, under no circumstances, 10 mg. 
It is possible that a single injection of 5 or 10 mg. may give more diagnostic 
results than repeated doses up to this amount, although it must be admitted 
that such large doses may react, in rare cases, positively in normal individuals. 

A positive result is very strong evidence of an active tubercular condition. 
A negative result is less reliable as some tubercular conditions, such as peri- 
tonitis or encapsulated foci elsewhere, may not react positively. 

Method of More. 

In this method use is made of a salve consisting of equal parts of old 
tuberculin and lanolin. A small bit of this salve is rubbed into the skin, pref- 
erably in the thoracic or abdominal region. After 24 to 48 hours small pale 
nodules, i to 2 mm. in diameter, are seen at the point of inoculation if the case 
be tubercular. A more severe reaction may be observed in the form of red- 

^ SeeHektoen, Jour. Am. Med. Assn., 191 2, LVIII, 1081; also, Vaughan, Am. Jour. Med. 
Sc, 1913, CXLV, 161. 

2 See Strickler and Goldberg, Jour. A. M. A., 1916, LXVI, 249; McBride and Schorer^ 
Ibid., 919; White, Jour. Cutan. Dis., 1916, XXXIV, 57; Strickler, N. Y. Med. Jour., 1916,. 
CIV, 198. 



664 DIAGNOSTIC METHODS 

dish miliary nodules. These nodules are limited to the point of application 
of the salve and disappear in 24 to 48 hours. Occasionally, some exudation 
may arise from the intense reactions, but this is very rare. This method is 
very simple and is very reliable.^ 

Method of von Pirquet. 

This method^ is very widely used, especially in cases of infection in children. 
The inner side of the forearm is cleansed with alcohol and ether and allowed to 
dry. Two drops of old tuberculin are placed on the skin at a distance of 6 to 
8 cm. from each other. The skin covered by these drops is then punctured by 
a large needle or gently scarified. The tuberculin is allowed to remain for 10 
to 15 minutes, when the clothing may be replaced. It is wise to make a third 
puncture or scarification between the two tuberculin points as a control upon 
the patient's reaction, no tuberculin being used here. A slight swelHng is 
usually produced which becomes surrounded by a light reddish border in a 
few hours, and disappears in 24 hours. If the reaction be positive, the area of 
puncture swells within 24 to 48 hours to a papule, reddish in color and 10 to 
15 mm. in diameter. The center may be pale and be surrounded with small 
serous points. By comparison with the normal point, a reaction may be 
more clearly demonstrated. The swelUng and redness begin to disappear 
within 48 hours, a slight pigmentation usually following this positive reaction. 
No general disturbance is noted as a rule. If the reaction be repeated, a marked 
positive result may be observed as an evidence of hypersensibility, the so-called 
anaphylactic reaction. This may, also, be noticed in the other methods, 
even though a much smaller dose be used than in the initial application. 

Method of Calmette. 

This method was advanced almost simultaneously by Calmette and Wolff- 
Eisner. One or two drops of a i to 100 dilution of old tuberculin in physio- 
logic salt solution are placed in the conjunctival sac, the lids being held apart for 
a few seconds to permit of absorption of the tuberculin. Only one eye is thus 
treated, the other being used as the control. A positive reaction appears in 
6 to 24 hours and may assume one of the following types: (i) Mere reddening 
of the caruncle and the inner surface of the lower lid; (2) the bulbar conjunc- 
tiva may take part in the process; (3) suppurative conjunctivitis with marked 
injection of the palpebral conjunctiva; (4) hemorrhagic conjunctivitis of 
the palpebral and bulbar portions with profuse fibrino-purulent exudation. 
An associated rise in temperature may be observed in the severe types. ^ 

^ See Krumbhaar and Musser, Am. Jour. Med. Sc, 1914, CXLVII, 540. 

^ See Wachenheim, Am. Jour. Dis. Child., 1912, IV, 27; Austrian, Jour. Exper. Med., 
191 2, XV, 149; Rosenberg, Ztschr. f. exper. Path. u. Therap., 1913, XII, 549; and Wachen- 
heim, Am. Jour. Dis. Child., 1913, V, 466; Klopstock, Ztschr. f. exper. Path. u. Therap., 

1914, XV, 13; Monrad, Ugesk. f. Laeger, 1914, LXXVI, 1419; KiichenhofF, Deutsch. med. 
Wchnschr., 1914, XL, 229; Salvetti, Pediatria, 1915, XXIII, 35; Fishberg, Arch. Pediat., 

1915, XXXII, 20; Am. Jour. Obs., 1915, LXXI, 361; Kraemer, Miinch. med. Wchnschr., 
i9i5,LXII, 5 and 46; Frazer, Med. Record, i9i5,LXXXVII, 57; Palmer, Jour.A.M.A.,1915, 
LXIV, 1312; Cattermole, Ibid., i9i5,LXV, 782; Manning and Knott, Am. Jour. Dis. Child., 
1915, X, 354; Heise, Am. Jour. Med. Sc, 1916, CLI, 862;Lindberg, Hygiea, i9i6,LXXVIII 
1553; Permin, Hospitalstid., 1916, LIX, 697; Weith, Rev. Med. de la Suisse Rom., 1916, 
XXXVI, 537; Craig, Jour. A. M. A., 1916, LXVII, 1227; Weil, Ibid., 1917, LXVIII, 
972; Cohen, Jour. Infect. Dis., 1917, XX, 233; Blechmann, Ann. de Med., 1919, VI, 200. 

2 See Vaughan, Jour. Am. Med. Assn., 1913, LXI, 1591. 



THE BLOOD 665 

Contraindications to this test are found in any inflammation of the eye or 
conjunctiva, in the previous treatment of the eye with tubercuHn, and recent 
application of tuberculin elsewhere. It would not seem that this method is 
advisable owing to the great discomfort which may arise and, especially, as it 
is not without danger of severe injury to the eye.^ 

Luetin Reaction. 

Noguchi^ has recently announced ''A Cutaneous Reaction in Syphilis," 
which may prove of great value in the diagnosis of this condition. His mate- 
rial is prepared as follows: Pure cultures of the treponema pallidum (see 
p. 640) are allowed to grow for periods of 6, 12, 24, and 50 days at 37°C. under 
anaerobic conditions. One was cultivated in ascitic fluid containing a piece of 
sterile placenta, and the other in ascitic fluid agar also containing placenta. 
The lower portion of each solid culture was cut out and the tissue removed. 
These agar columns, containing large numbers of spirochetae, were carefully 
ground in a sterile mortar. This paste was then gradually diluted by adding, 
little by little, the fluid culture, until the emulsion became perfectly liquid. 
This mixture was heated to 6o°C. for one hour in a water-bath and 0.5 per cent, 
carbolic acid was added. When this mixture is examined under the dark-field 
microscope 40 to 100 pallidae may be seen in each field. This suspension is 
called luetin.^ 

The skin of the upper arm is sterilized with alcoholic sublimate solution 
before the injection. The amount of luetin injected is 0.05 c.c. to 0.07 c.c. 
This injection is intradermic, that is, in the skin as superficially as possible. 
In normal cases there appears, after 24 hours, a very small erythematous area 
at and around the point of injection. No pain or itching is experienced. This 
slight reaction gradually recedes within 48 hours and leaves no induration. 
In certain cases the reaction may reach a stage of small papule formation after 
24 or 48 hours, after which time it commences to recede. 

In positive cases the following types of reaction occur : 

{a) Papular Form. — A large, raised, reddish, indurated papule, usually 5 
to 10 mm. in diameter, makes its appearance in 24 to 48 hours. The papule 
may be surrounded by a diffuse zone of redness and show marked telangiec- 
tasis. The dimensions and the degree of induration slowly increase during the 
following three or four days, after which the inflammatory processes begin to 
recede. The color of the papule gradually becomes dark bluish-red. The 
induration disappears within a week, except in certain instances in which a 
trace of the reaction may persist for a longer period. This latter effect is usu- 
ally met with among cases of secondary syphilis under regular mercurial treat- 
ment in which there are no manifest lesions at the time of making the skin 
test. Cases of congenital syphilis also show this reaction. 

^Austrian (Bull. Johns Hopkins Hosp., 1911 XXIII, i) advocates a similar test for 
typhoid fever. The antigen is a "typho-protein" prepared from a mixed culture of 80 
different typhoid strains. Positive results are obtained in 95 per cent, of cases of typhoid 
while it is negative without exception in practically all controls. Its great advantages are 
that it is a bedside test and is given in the early stages of the disease. 

2 Jour. Exper. Med., 1911, XIV, 557; Munch, med. Wchnschr., 1911, LVIII, 2372. 

^Luetin may now be obtained either from Parke, Davis and Co. or Mulford Co. 



666 DIAGNOSTIC METHODS 

{B) Pustular Form. — The beginning and course of this reaction resemble 
the papular form until about the fourth or fifth day, when the inflammatory 
processes commence to progress. The surface of the indurated round papule 
becomes mildly edematous, and multiple miliaty vesicles occasionally form. 
At the same time a beginning central softening of the papule obtains. Within 
the next 24 hours, the papule changes into a vesicle filled at first with a 
semi-opaque serum that later becomes definitely purulent. Soon the pustule 
ruptures. The margin of the broken pustule remains indurated, while a crust 
quickly forms, which falls off within a few days. The induration soon disap- 
pears leaving almost no scar. This reaction was found almost constantly 
in tertiary types as well as in secondary or hereditary forms which had been 
treated with salvarsan. 

(C) Torpid Form. — In rare instances, the injection sites fade away to 
almost invisible points within three or four days, so that they may be passed 
over as negative reactions. Sometimes these spots suddenly light up again 
after 10 days or so and progress to small pustular formations. 

No marked constitutional symptoms have been observed after the use of 
the luetin. In most positive cases a slight rise in temperature took place 
lasting for one day. 

The conclusions of Noguchi,^ based upon a study of 400 cases, are as 
follows: Luetin produces a cutaneous reaction in syphilitic and parasyphilitic 
patients that is most constant and severe in the tertiary and hereditary affec- 
tions. During the primary and secondary stages, the reaction is infrequent, 
and when present is of mild degree. An exception has been found in cases in 
which energetic treatment has been or is being carried out and in which clin- 
ical signs of s>TDhilis are absent. ^ Such cases may show a severe reaction, 
especially those treated with salvarsan. In certain cases of old infection in 
which no treatment has been taken and in which no symptoms have appeared 

1 See Ziegel, Arch. Int. Med., 191 2, IX, 520; Miiller and Stein, Wien. klin. Wchnschr., 

1913, XXVI, 408; Klausner, Ibid., 973; Noguchi, Presse med., 1913, XXI, 757; WoKsohn, 
Jour. Am. Med. Assn., 1913, LX, 1855; Kaliski, New York Med. Jour., 1913, XCVIII, 24; 
Schmitter, Jour. Cutan. Dis.; 1913, XXXI, 549; Brown, Am. Jour. Dis. Child., 1913, VI, 
171; Foster, Am. Jour. Med. Sc, 1913, CXLVI, 645; FagiuoH and Fisichella, Berl. klin. 
Wchnschr., 1913, L, 181 1; Gavini, Riforma Med., 1913, XXIX, 985, 1013, 1049, 3-iid 1075; 
McNeil, Jour. Am. Med. Assn., 1914, LXII, 529; Joltrain, Ann. de Med., 1914, I, 337; 
Fagiuoli and Fisichella, Berl. klin. Wchnschr., 19 14, LI, 449; Kilgore, Jour. Am. Med. 
Assn., 1914, LXII, 1236; Much, Med. Klin., 1914, X, 811; Blechmann and Delort, Ann. de 
Med. et Chir. Infant., 1914, XVIII, 406; Blechmann, Delort and Tulasne, Ibid., 465; 
Cannata, Pediatria, 1914, XXII, 481; Pusey and Stillians, Jour. Cutan. Dis., 1914, XXXII, 
560; Noguchi, New York Med. Jour.. 1914, C, 349; Muscel, Dersca and Friedmann, 
Munch, med. Wchnschr., 1914. LXI, 1272; Clausz, Ibid., 1933; Curti, Rif. med., 1914, 
XXX, 1264; Boas and Stlirup, Hospitalstid., 1914, LVII, 1417; Trossarello, Gazz. d. osp., 

1914, XXXV, 457; Wolff and Zeeman, Ibid., 811; Schippers, Ibid., 817; Kafka, Berl. klin. 
Wchnschr., 1915, LII, 15; Ross, Jour. Ment. Sc, 1915, LXI, 244; Sherrick, Jour. Am. Med. 

1915, LXI, 244; Sherrick, Jour. Am. Med. Assn., 1915, LXV, 404; Hanes, Am. Jour. Med. 
Sc, 1915, CL, 703; Kolmer and Broadwell, Jour. ImnunoL, 1916, I, 429; Fletcher, Lancet, 

1916, II, 710; Fulton and Cummings, Am. Jour. Syph., 1917, I, 663; Cole and Paryzek, 
Jour. A. M. A., 1917, LXVIII, 1089. _ 

2 As this luetin reaction is an allergic one, it is not surprising to find that, in the primary 
and secondary stages, there are not sufi6cient antibodies present to produce the reaction. 
Although emulsion of ascitic agar itself may produce an intradermic reaction similar to that 
of luetin, yet this does not militate against the test as it shows a hypersensitiveness, which is 
itself rather characteristic of syphilis. See Stokes ,Jour. Infect. Dis., 1916, XVIII, 415; 
Jour. A. M. A., 1917, LXVIII, 1092. 



THE BLOOD 667 

for many years, and in the course of which miscarriages have occurred, this 
reaction has failed to appear. Despite the absence of symptoms, mothers 
who have young syphiHtic children have usually given the reaction. It re- 
mains to be determined in how far this reaction can be used to supplement 
the Wassermann reaction. It appears probable that the Wassermann reac- 
tion is more constant in the primary and secondary, and the cutaneous reaction 
in the tertiary and latent forms of syphilis.^ It further appears that the 
Wassermann reaction is more directly and immediately affected by anti- 
syphilitic treatment than is the cutaneous reaction. ^ 

Schick Reaction. 

By means of this reaction individual susceptibility and immunity to diph- 
theria may be readily recognized in a great majority of cases. It consists in 
the intracutaneous injection of diphtheria toxin of definite dosage and the 
observance of the irritant action at the point of injection. 

Technic. 

In the first place the minimum fatal dose of the diphtheria toxin is deter- 
mined for a guinea-pig of 250 grams weight.^ This amount is then so diluted 
with sterile physiologic salt solution that each c.c. of the dilution contains 0.2 
of the minimum fatal dose. Of this diluton o.i c.c. (representing one-fiftieth 
of the minimum fatal dose) is injected into the skin by means of an accurately 
graduated syringe (i c.c. record tuberculin syringe) with steel or platinum- 
iridium needle, which should be very fine, sharp and she rt-pointed. The site 
of injection is the skin of the outer side of the upper portion of the arm. 

Reaction. 

If the injection be properly done, one observes a white blister-like punctate- 
appearing wheal, which shows distinct markings of the openings of the hair 

1 See Robinson, Jour. Cutan. Dis., 191 2, XXX, 410. Sherrick (Jour. Am. Med. Assn., 

1 91 5, LXV, 404) calls attention to the fact that recent or simultaneous administration of 
potassium iodid will cause a positive luetin reaction in non-syphilitic individuals. Kolmer, 
Matsunami, and Broadwell, Jour. A. M. A., 191 6, LXVII, 718. 

2 Irons has recently perfected a local skin reaction in cases of generalized gonorrheal 
infections, using as his vaccine a glycerine extract of autolyzed gonococci. After a few 
hours a papule is formed with a surrounding area of hyperemia, the maximum reaction being 
reached in 24 hours. Irons classifies as positive the reactions showing a total diameter of 5 
mm. or over; negative when 3 mm. or less (Jour. Infect. Dis., 191 2, XI, 77). See, also, 
Fronstein, Med. Obozr., 1913, LXXIX, 225. 

Gay and Force (Arch. Int. Med., 1914, XIII, 471) have introduced a skin reaction for 
typhoid fever, which consists in using a "typhoidin" solution, prepared by concentrating a 
glycerin bouillon culture of t5^phoid bacUli, and which is applied to the abraded skin. A 
difference of 2.5 mm. between the areolae of the "typhoidin" point and the control point is 
taken as evidence of a positive reaction. See, also, Gay, Am. Jour. Med. Sc, 1915, CXIX, 
157. Pulay, Wien. klin. Wchnschr., 1915, XXVIII, 1189; Kolmer and Berge, Jour. Immunol., 

1916, I, 409; Austrian and Bloomfield, Arch., Int. Med., 1916, XVII, 663; Kilgore, Ibid., 
25; 1917, XIX, 263 and 276; Force and Stevens, Ibid., 440; Gay and Lamb, Jour. Lab. and 
Clin. Med., 1917, II, 217; Meyer and Christiansen, Jour. Infect. Dis., 1917, XX, 357, 391 
and 424. 

^ This toxin may be obtained from the larger laboratories handling biological products or 
may be procured from the health oJS&cers in the larger cities. In this latter case a definite 
amount of a known potency toxin is supplied together with a diluent fluid. After the toxin 
is diluted it deteriorates rapidly, so that it should not be used after 12 hours' standing. 
The undiluted toxin, obtained from the supply houses should be kept cool to prevent 
deterioration. Usually the potency of the toxin is stated on the package, so that the neces- 
sary dilution may be directly figured. 



668 DIAGNOSTIC METHODS 

follicles. This elevation (due to the traumatism) persists for a short time, all 
signs of it usually disappearing within an hour. If there is sufficient antitoxin 
in the blood of the subject to neutralize the small amount of toxin, no further 
changes should be observed. This is a negative reaction and indicates that 
the person is immune to diphtheria. 

A positive reaction is noted in the gradually increasing redness and infil- 
tration of the skin, a distinct circumscribed area of redness and infiltration 
measuring about i to 2.5 cm. being seen within 24 or 48 hours. This redness 
persists for several days and gradually fades, leaving a brownish pigmenta- 
tion and slight scaling. 

In some cases, especially in older children and adults, a pseudo-reaction is 
observed, which is not due to the action of the toxin but is, rather, an ana- 
phylactic response to the protein substance of the diphtheria bacillus or, as 
Kolmer and Moshage believe, to trauma of a skin which is unduly sensitive. 
Some local redness and infiltration may be noted, but this comes on more 
rapidly than does the true reaction, the redness is less sharply outlined, the 
local infiltration is more marked and the signs disappear in 2 to 4 days with 
little or no pigmentation and no scaling. These false reactions are confusing, 
but should be easily differentiated by careful observation. To control the 
reaction, one may inject into the other arm o.i c.c. of bouillon diluted i to 10 
or I to 1,000. An extreme sensibility of the skin will be indicated by slight 
local reaction in the control arm. 

Significance. 

In normal subjects a negative result indicates, in practically 100 per cent, of 
cases, the presence in the blood of diphtheria antitoxin and, hence, an im- 
munity to diphtheria. A typical positive reaction points to an absence of anti- 
toxin and a resulting susceptibility to infection. 

From the reports of the literature, it is evident that about 80 per cent, of 
new-born children, 50 to 60 per cent, of children, especially those between the 
ages of I and 5, and 90 per cent, of adults contain sufficient antitoxin, as 
shown by this test, to make them immune to diphtheria. In the presence of 
diphtheria, the question of exposure of other children is an important one. It 
is evident that immunization by injection of antitoxin is indicated only in 
those giving positive Schick reactions. Further, by means of this test definite 
separation of the diphtheria cases from non-diphtheritic cases may be made, 
as patients with true diphtheria have little antitoxin in the blood and hence 
show a positive reaction. Likewise diphtheria carriers may be separated 
from actual diphtheritics, as the former usually have a large amount of anti- 
toxin in the blood and, therefore, give negative reactions. 

There can be little question but that this test will prove invaluable in 
hospitals in selecting internes and nurses to look after the diphtheria patients. 
Moreover, the question of isolation of actual diphtheria patients becomes a 
much easier matter, with the knowledge that those showing a negative Schick 
reaction are immune to diphtheria and need not, therefore, be removed from 



THE BLOOD 669 

chance of infection. The economic as well as the diagnostic value of this 
test seems to be very great. ^ 



VIII. Sero-Diagnosis 

In the attempt to enlarge our diagnostic procedures advantage has been 
taken of many of the principles previously discussed. The methods evolved 
are among those most frequently employed and are reliable within certain 
small limits which will be mentioned later. They are all based upon certain 
peculiar properties possessed by the serum of patients with infectious diseases 
and are, therefore, of special importance in differential diagnosis. 

A . Agglutination Reactions. 

Since the work of Gruber and Durham and of Pfeiffer and Kolle in 1896, 
the fact has been well established that the serum of an animal, which has been 
rendered immune to certain bacteria, shows the property of agglutinating or 




Fig. 154. — Bacillus typhosus at beginning of Widal test. (Da Costa.) 

clumping homologous bacteria, owing to the production of immune bodies, 
the so-called agglutinins. This reaction takes place in high dilutions of the 
serum and is practically specific for any given organism. Widal, ^ in the same 

1 Schick, Miinch. med. Wchnschr., 1913, LX, 2608; Groer and Kassowitz, Ztschr.^f. 
Immunitatsf. u. exper. Therap., 1914, XXIII, 108; Kassowitz and Schick, Ztschr. f. d. ges. 
exper. Med., 1914, II, 305; Otto, Deutsch. med. Wchnschr., 1914, XL, 542; Schick, Kasso- 
witz and Busacchi Ztschr. f. d. ges. exper. Med., 1914, IV, 83; Park, Zingher and Serota, 
Arch. Pediat., 1914, XXXI, 481; Jour. Am. Med. Assn., 1914, LXIII, 859; Veeder, Am. 
Jour. Dis. Child., 1914, VIII, 154; Kolmer and Moshage, Ibid., 1915, IX, 189; Weaver and 
Maher, Jour. Infect. Dis., 1915, XVI, 342; Bundesen, Jour. Am. Med. Assn., 1915, LXIV, 
1203; Graef and Ginsberg, Ibid., 1205; Moody, Ibid., 1206; Kolmer and Moshage, Ibid., 
1915, LXV, 144; Zingher, Ibid., 329; Moffett and Conrad, Ibid., loio; Birnberg, St. Paul 
Med. Jour., 191^, XVII, 204;LevinsonandBlatt, Arch. Diag., 1915, VIII, 201; Zuckermann, 
New York Med. Jour., i9i5,CII, 808; Weaver and Maher, Jour. Infect. Dis., 1915, XVI, 342; 
Park and Zingher, Jour. A. M. A., i9i5,LXV, 2 2i6;Levinson, 111. Med. Jour., 1915, XXVIII, 
405; Sprenger, Ibid., 1916, XXIX, 447; Koplik and Unger, Jour. A. M. A., 1916, LXVI, 
1 195; Bessau and Schwenke, Monatsschr. f. Kinderhkde., 1916, XIII, 393; Kolmer, Jour. 
Immunol., 1916, I, 443; Bullen, N. Y. State Jour. Med., 1916, XVI, 208; Griswold, Jour. 
Lab. and Clin. Med., 1916, I, 441; Weaver and Rappaport, Jour. A. M. A., 1916, LXVI, 
1448; Zingher, Ibid., 161 7; Am. Jour. Dis. Child., 1916, XI, 269; Ibid., 1917, XIII, 247; 
Arch. Int. Med., 1917, XX, 392. 

2 Bull. med. Paris, 1896, X, 618 and 766. 



670 



DIAGNOSTIC METHODS 



year, applied this principle to the diagnosis of typhoid fever, showing that 
clumping and loss of motility of the typhoid bacilli occur when a suspension 
of the actively motile types is treated with an homologous immune serum at 
such a dilution that normal or non-homologous serum does not react. 
I. Gruber-Widal Test. 




Fig. 155. — A pseudo-Widal reaction. {Da Costa.) 

Cultures. 

The cultures of the typhoid bacillus must be fresh and must show^ many 
actively motile organisms in the hanging-drop specimens, if they are to be 
used in the microscopic method. It is advisable always to use cultures con- 
taining about the same number of bacilli, as the reaction is somewhat quan- 
titative. Thus, if few bacilli be present, they may be clumped by a small 




Fig. 156. — A positive Widal reaction. {Da Costa.) 

amount of agglutinin both specific and non-specific, while if the number be 
very large, the specific agglutinin may not be sufl&cient to cause marked clump- 
ing and loss of motiUty of the organisms. Great differences toward aggluti- 
nation exist in different strains of the typhoid bacillus, so that it is essential 
always to employ, for diagnostic purposes, strains which have passed 
through several generations on artificial media. 

A stock culture of the bacillus is kept in sealed tubes of nutrient agar in a 
cool dark place and, from this stock, fresh agar cultures are made every few 



THE BLOOD 67 I 

weeks as occasion may require. From these stock agar cultures, bouillon 
tubes are inoculated and incubated for 15 to 24 hours. These latter are used 
for the agglutination tests. These bouillon cultures should be equally cloudy 
throughout and show no pellicle formation or gross clots of bacilli. It is wise 
to make up fresh bouillon cultures every other day so as to be certain of the 
maximum motility of the organisms.^ 

In the use of the macroscopic method cultures killed by heat, formalin, 
carbolic acid, thymol, etc., may be employed. Many laboratories through- 
out the country supply these cultures for general work. Picker's "Typhus 
diagnostikum " is one of the widely used types. For self-evident reasons it is 
wise to have some standard strength of these cultures. Hastings advocates 
a mixture of 5 c.c. of 5 per cent, carbolic acid solution, 10 c.c. of glycerin, and 
85 c.c. of physiologic salt solution to which are added the organisms scraped 
from the surface of two 24-hour agar-slant cultures of bacillus typhosus (the 
bacilli being rubbed into the mixture with a spatula). Bass advises a sus- 
pension of 10,000 million dead typhoid bacilli per c.c. in 1.7 per cent. NaCl 
solution to which i per cent, formalin is added. 

Obtaining the Blood. 

In this procedure much depends upon the custom of the worker as to the 
method followed. As this reaction is a pure serum one, it is much more logical 
to use the serum than the whole blood, if one has any choice in the matter. 

The simplest and best clinical method in the use of a small capillary tube 
(made from J^-inch glass tubing) with a central bulbar enlargement into 
which the blood (15 to 20 drops) is drawn by capillarity from a puncture of 
the ear. The tube is then laid flat until coagulation occurs and the serum 
separates. To hasten this process, the end of the tube, which contains no 
blood, may be sealed in the flame and the tube centrifuged. If it is desired 
to send the specimen to the laboratory, seal both ends and pack in such a 
way as to insure it against breakage. In this connection it may be well to 
recall that Lyons ^ has shown the possibility of obtaining a culture of the 
bacillus typhosus from the clot settling in such tubes. This may be done 
by the method previously outlined. Another method of obtaining the serum 
is to apply a cantharides blister which will furnish enough serum in 6 to 
12 hours for many tests. Venous puncture may, also, be employed, but 
this is rarely advisable as sufficient serum may be more easily obtained in 
the above ways. 

Some workers use the whole blood instead of the serum. If this be done, 
the leucocytometer may be employed, using physiologic salt (0.9 per cent.) 
solution as the diluent. Draw the blood from a puncture in the ear to the 
mark i and the diluent to 10. This gives a dilution of the whole blood of i 
to 10 or I to 20 of the plasma, as the plasma forms approximately 50 per cent. 

^ Riemer (Miinch. med. Wchnschr., 1913, LX, 908) calls attention to the fact that 
typhoid bacilli show diminished agglutination if grown in alkaline media. See, also, Gay 
and Claypole, Arch. Int. Med., 1913, XII, 621; Jour. Am. Med. Assn., 1913, LX, 1141; 
Bull and Pritchett, Jour. Exper. Med., 1916, XXIV, 35. 

2 Arch. Int. Med., 1909, IV, 64. 



f 



672 DIAGNOSTIC METHODS 

of the whole blood. This dilution may then be used directly for the later 
work by mixing with an equal volume of the typhoid suspension. 

More frequent, possibly, in the hands of workers in municipal or the larger 
clinical laboratories, is the employment of dried blood sent to them on glass 
slides, bits of paper, mica, etc. Such specimens may be easily transported 
but can not yield anything but an approximate result, as an accurate dilution 
is out of the question unless very careful weighing of the blood is carried out. 
A large drop of the dried blood is dissolved in 10 drops of water, the dilution 
being called i to 10. If the blood be upon paper, the dilution must be made 
largely by guess, experience teaching an approximate dilution from the color 
of the mixture. These dilutions will, of course, be doubled when the test 
is applied. 

Dilution of the Serum. 

The methods of diluting the whole fresh blood and the dried blood have 
already been given. As diluting pipets for the serum, one uses small ( J^ inch) 
glass tubing which has been drawn into a long capillary at one end. A rubber 
bulb fits over the larger end and provides the suction. It is good practice 
always to have a number of these pipets on hand so that they may be dis- 
carded or thoroughly cleaned before a second use. If the same pipet be 
employed for different cases, a contamination may arise to such an extent 
that erroneous results obtain. 

A few drops of the serum, collected as above, are drawn into the capillary 
tube by suction. From this tube the serum is then dropped into the mixing 
vessels, preferably watch crystals. Two dilutions are usually made in the 
writer's laboratory, namely i to 25 and i to 50. Allow i drop of serum to fall 
into each of two watch crystals, the remaining serum in the capillary being 
blown into the original collecting tube. Now add, from the same pipet used 
in dropping the serum in order to insure drops of the same size, to one of 
these drops of serum 24 drops of physiologic salt solution and to the other 49 
drops of the same diluent. Mixtures of these diluted sera with an equal 
volume of typhoid suspension, as employed in the test, give final dilutions of 
I to 50 and I to 100. 

Microscopic Reaction. 

The serum (preferably), whole blood, or dried blood is diluted as previously 
described. Mixtures are then made of these dilutions with an equal 
volume of typhoid suspension. By means of a platinum loop of stiff wire, a 
loopful of the diluted serum {1 to 25) is placed on the center of a cover-slip. 
After burning the serum from the loop, a loopful of the typhoid suspension is 
transferred to the same cover-glass and the two fluids thoroughily mixed by 
means of the loop. The dilution of the serum will then, evidently, be double 
its original dilution, or i to 50. The ordinary hanging-drop preparation is 
then made by inverting the cover-slip over the hollow of the slide, which has 
been ringed with vaseline. Instead of using the hanging-drop method, some 
prefer to place the cover directly upon the slide. Usually the specimen 
does not dry sufficiently to introduce errors, but the results are certainly not 
as characteristic by this procedure in all cases. 



THE BLOOD 673 

The specimen is best studied with the high-power lens, using a strong 
light. The organisms will be seen, in specimens properly lighted, as free 
actively motile bacilli moving with their rapid darting motion through the 
field. In the course of 45 to 60 minutes, or often in much less time, the organ- 
isms will be seen to be collected in clumps and to be motionless, except for a 
slight vibratory movement. This is a typical positive reaction, namely 
marked clumping with complete loss of motility in one hour at a dilution of i 
to 50. Such a reaction is practically never due to any other disease than 
typhoid fever, if the culture of the bacillus be a pure one. 

In some cases marked agglutination with incomplete loss of motility 
obtain. In the writer's opinion this can be interpreted only as a doubtful or 
incomplete reaction. The probability is that typhoid fever is present, but 
one must admit the possibility of group agglutinins, due to the paratyphoid 
bacillus, as well as agglutinins of other infections. When dried blood is used, 
a slight tendency to agglutination may be observed, due to the presence of 
small particles of fibrin, so that in such cases the loss of motility should be 
demanded even more rigorously than when serum is used, before a positive 
result is reported. It has been shown that the serum of many healthy patients 
or that of subjects suffering with diseases other than typhoid fever frequently 
show a reaction similar to that described above. This statement is true only 
when the question of dilution of the serum is taken into account. The char- 
acteristic Widal reaction is shown at a dilution of i to 50 or higher, while in 
other diseases, with the exception of paratyphoid, the reaction practically 
disappears (the typhoid bacillus being, of course, used) at a dilution of i to 
20. The element of time is also of great importance. In typhoid fever, the 
reaction may occur instantly at i to 10 or 20, and in one-half to one hour at 
I to 50, yet a period ranging from one-half to one hour is necessary with other 
sera at a dilution of i to 10 or 20. On the other hand, there are cases of 
undoubted typhoid fever that show no agglutination at i to 10 or 20, while at 
I to 50 this is marked. What occurs here is uncertain, some crediting the 
paradox to bacteriolysis of the organisms at the former dilutions. 

Macroscopic Reaction. 

The method is based on the fact that the agglutination of typhoid bacilli by 
a potent homologous serum is visible to the naked eye, the clumps of bacilli 
settling to the bottom of the test-tube leaving a clear supernatant fluid. 

If living cultures of bacillus typhosus are employed, the best technic is the 
following: Make up the serum dilutions as previously described, using i 
to 50 and I to loo dilutions. To these diluted sera, contained in small sterile 
test-tubes (ii cm. long by 88 mm. wide), is added a loopful of typhoid bacilli 
scraped from the surface of a 24-hour agar culture. The loop is rubbed 
against the inner surface of the test-tube until the bacteria have been sus- 
pended in the serum. The turbid suspension is then thoroughly shaken until 
it becomes uniformly cloudy, no clumps of bacteria being seen, and is placed 
in the incubator at 37°C. for one hour. The tubes are then removed and 
examined by strong transmitted light. The agglutinated bacteria may easily 
be recognized by their granular fiocculent appearance. General clumping 
43 



674 DIAGNOSTIC METHODS 

should obtain in one hour at a dilution of i to 50, while complete precipitation 
(settling) should follow in 24 hours, a clear supernatant fluid being left be- 
hind. This is the positive reaction. A negative result is shown by no clump- 
ing and little, if any, loss in the turbidity of the mixture. 

For the general practitioner, who desires to use the macroscopic method, 
the employment of cultures killed by heat or chemical means is recommended, 
as it obviates the necessity of an incubator, culture media, or microscope and 
permits him to have on hand material for his tests. Any of the suspensions of 
killed organisms may be used, Ficker's and Hastings' being widely employed. 
Dilute the serum as previously described, the dilutions in this case being i to 
25 and I to 50. To this diluted serum (3^ c.c), contained in the small test- 
tubes, add an equal volume of the suspension of killed bacteria and examine 
as above mentioned. The clumping should be observed in one hour and 
complete clearing should obtain in 24 hours, if the reaction be positive. 
This method gives very reliable results and is very simple. 

Method of Bass and Watkins. 

These workers^ have introduced a modification of this macroscopic agglu- 
tination test which is simple, reliable, and quick. It has the advantage that 
it can be carried out at the bedside, the result being known in 2 to 3 minutes. 
Little equipment is required. The suspension consists of 10,000 million 
killed typhoid organisms per c.c. in 1.7 per cent. NaCl solution to which i per 
cent, formalin is added. 

Allow a full drop of blood to fall into 4 drops of water and mix thoroughly. 
Instead of this, one may make a blood-smear (using approximately J^ drop of 
blood) and dissolve this on the slide with i drop of water, the mixture being 
stirred with a tooth-pick or similar substance. With this diluted blood (i to 4) 
mix an equal amount of the above suspension of typhoid bacilli on a glass slide. 
Tilt the slide from side to side so as to keep the mixture flowing back and 
forth. If the reaction is positive, a grayish mealy sediment appears within 
one minute, usually in less time. This sediment appears in the fluid around 
the edges and tends to collect there. If the agitation is continued, the clumps 
increase in size for 2 to 3 minutes. If the reaction does not appear in this 
time, it will not show up at all. When the reaction is negative, no agglu- 
tination occurs and the mixture remains as clear and unchanged as when 
placed on the slide. 

Recently Michaelis^ has advocated a new agglutination reaction for the 
bacillus typhosus as well as other bacteria. This is based on the fact that 
bacteria are agglutinated by acids, the rate and degree being dependent upon 
the concentration of hydrogen ions present. This test promises much in the 
future as it depends entirely upon chemical factors which are more easily and 
more exactly controlled than are the uncertain biologic units with which we 
usually work.^ 

1 Arch. Int. Med., 1910, VI, 717. 

2 Folia Serologica, 191 1, VII, loio; Deutsch. med. Wchnschr., 1915, XLI, 241. 

3 See Beniasch, Zeitsch. f. Immun.-Forsch., 191 2, XII, 268; Gillespie, Jour. Exper. 
Med., 1914, XIX, 28. 



THE BLOOD 675 

Time of Appearance and Disappearance of the Reaction. 

The time at which a Widal reaction may appear in the course of a typhoid 
-is of some importance. The earhest date of appearance of a positive reaction 
is rather hard to determine, as workers differ in their interpretation of the 
beginning of the disease. Definite reports of positive results as early as the 
second day have been given by Fraenkel and others. Although this reaction 
may appear before the rose spots, splenic tumor, or diazo reaction, yet it is 
practically never found when the blood shows a normal or an increased num- 
ber of leucocytes, a leucopenia with a relative lymphocytosis being associated 
with a positive Widal in practically allcases. A differential blood count may, 
therefore, be of great value when the means of making an agglutination test are 
not at hand. Wood was able to obtain positive results in only lo per cent, of 
his cases in the first week of the disease. The majority of observers are 
agreed that the first appearance of this reaction is in the second week of the 
disease in most cases. In a certain number, it may not appear till the third or 
fourth week the highest agglutinations frequently being seen toward the end 
of the disease and in the early stages of convalescence. It is very rare not to 
obtain a positive reaction at some stage of the disease, providing the tests 
are made at different intervals. It is possible that cases reported, in which no 
Widal reaction was observed throughout the course of the disease, may be 
due to faulty technic or to insufficient tests. A single negative test, of course, 
proves nothing. 

The persistence of the Widal reaction is likewise variable. Little seems to 
depend upon the severity of the attack as to the time of disappearance of the 
reaction. Usually this reaction persists for i to 2 months, although it 
may obtain for 4 to 6 months or even for years. It is undetermined 
whether these latter cases are due to reinfection rather than a continuation of 
the agglutination powers of the serum. Reports are extant of cholelithiasis 
in which typhoid bacilli were isolated from the gall-bladder as long as 18 
years after an attack of typhoid, the Widal reaction being positive in these 
cases. In this connection it is to be said that "typhoid-carriers" do not 
show a positive Widal as a rule. 

Specificity of the Reaction. 

From the standpoint of the biologic theory, the typhoid bacillus is aggluti- 
nated only by an homologous immune serum. Practically, we find that this 
is the case within certain limits, if all conditions are properly met. A posi- 
tive Widal reaction is not infrequently met with in cases of jaundice, in 
which no specific disease is clinically evident.^ However, it is certain that 
most, if not all, of these cases of jaundice are actually secondary to a typhoid 
cholecystitis with its accompanying sequelae. Such patients may have an 
"ambulatory" typhoid or may have no clinical signs other than a positive 
Widal. 

^ See Mazza, Gazz. d. osp., 1914, XXXV, 921; Perlmann, Munch, med. Wchnschr., 1915, 
LXII, 435; Stuber, Ibid., 11 73; Bernard and Paraf. Presse med., 1915, XXIII, S33'y Plass- 
berg, Wien. klin. Wchnschr., 1915, XXVIII, 1314; [Davison, Jour. A. M. A., 1916, LXVI, 
1297; Pollak, Wien. klin. Wchnschr., 1916, XXIX, 1204; Baerthlein, Miinch. med. Wchn- 
schr., 1916, LXIII, 1564; O'Farrell, Lancet, 1916, II, 970; Lev}'- and Vallery-Radot, Ann. 
de Med., 1916, III, 504 and 515. 



676 



DIAGNOSTIC METHODS 



Further, in many cases of infection with bacteria of the colon group, a 
^'collateral agglutination" may obtain, that is agglutination of several 
members of the group may occur with one serum. Cases are undoubtedly 
met with in which the infection is due to the paratyphoid bacillus and in 
which the agglutination of the typhoid bacillus is stronger than the para- 
typhoid; while, on the other hand, cases of true typhoid may show a stronger 
reaction with bacillus paratyphosus than with the bacillus typhosus. Accord- 
ing to Lentz, the time of such associated agglutinations is important in this 
connection. The paratyphoid bacillus is agglutinated more quickly by a 
paratyphoid serum than is the typhoid bacillus by the typhoid serum. 

A positive reaction, all conditions outlined above obtaining, indicates 
infection with the bacillus typhosus in practically all cases. This test can not 
be regarded as of great value in the early stages, as only a very small percent- 
age of cases show early agglutination reactions. It is especially serviceable 
in the differential diagnosis from the second week of the disease on to convales- 
cence. In the ambulatory types or in localized typhoid infections, especially 
in the gall-bladder, this test is of great value. A negative reaction should 
not be interpreted as excluding typhoid, as many typical and severe cases 
show no reaction until late in the disease.^ Like all laboratory tests, it must 
be used only in connection with the clinical findings, if the best results are to 
follow. The attending physician is the one to interpret the result, not the 
laboratory worker. 

2. Diseases Other than Typhoid Fever. 

Other organisms, beside the bacillus typhosus, show such agglutination re- 
actions. The technic is the same and the results comparable, the same 
specificity being shown. It is especially in the realm of infections with 
members of the colon group that these tests are, frequently, uncertain, 
yet, here also, careful variation in the dilution of the serum and observation 
of the time and degree of agglutination will lead to a positive result, which 
will be of the greatest clinical value. 

The organisms, frequently identified by these reactions, are the para- 
typhoid bacilli, cholera spirillum, Shiga bacillus, bacillus of plague, malta- 
fever, tuberculosis, pertussis, influenza, and the diplococcus of pneumonia. ^ 

^ The widespread use of typhoid vaccine as a preventive inoculation has introduced a 
large possible error into this reaction. The serum of injected (immune) patients shows a 
positive Widal reaction very quickly. This condition persists for a varying period, so that 
blood cultures or Austrian's ophthalmo-reaction (see p. 665) should be used in such cases. 
See Moon, Jour. Am. Med. Assn., 1913, LX, 1764; also Kellermann, Mlinch. med. Wchn- 
schr., 1914, LXI, 2453; Hirschbruch, Deutsch., Med. Wchnschr., 1915, XLI, 525; Hamil- 
ton, Jour. A. M' A., 1915, LXV, 1873; Tonnel, Lyon Med., 1916, CXXV, 105; Chante- 
messe, Bull de I'Acad. Med., 1916, LXXVI, 140; Gautier and Weissenbach, Presse Med. 
1916,'XXIV, 413; Walker, Lancet, 1916, II, 896; Rist, Ann.de Med., 1916,111,54; Salomon 
Presse Med., 1916, XXIV, 91; Conradi and Bieling, Deutsch. Med. Wchnschr., 1916, 
XLII, 1280; Danila and Stroe, C. R. soc. biol. Paris, 1916, LXXIX, 108; Meyer and Kil- 
gore. Arch. Int. Med., 191 7, XIX, 293; Rist, Jour. Lab. and Clin. Med., 1917, III, i; 
Klemperer and Rosenthal, Ztschr. f. klin. Med., 1918, LXXXVI, i; Krumbhaar and 
Smith, Jour. Infect. Dis., 1918, XXIII, 126. 

2 Kolmer (Jour. Exper. Med., 191 3, XVIII, 18) has shown that there is no appreciable 
amount of agglutinin for treponema pallidum in the sera of secondary and tertiary syphilis. 
See, however, Zinsser and Hopkins (Jour. Exper. Med., 1915, XXI, 576, and Kissmeyer, 
Deutsch. med. Wchnschr., 1915, XLI, 306. 



THE BLOOD * 677 

B. Precipitin Reaction. 

This reaction is based upon another fact well established in serum path- 
ology. It has been proven that the serum of an animal, injected with blood 
or blood-serum of another animal, shows the property, when added to an 
homologous serum, of precipitating the albumin of this serum in the form of a 
Hght fiocculent precipitate. This same pecuHarity has been observed after 
injection of exudates or transudates or of pure proteins both animal and vege- 
table.^ These reactions are, therefore, specific for the type of protein injected, 
within certain definite limits. Although this test has been used in the 
detection of certain albuminous substances excreted in the urine (see p. 276), 
yet its chief interest to the laboratory worker is its application to the medico- 
legal detection of blood and blood-stains. ^ 

The work of Wassermann and Uhlenhuth has shown that blood-stains 
may reveal their origin from any particular species of animal by application 
of this test. It is evident that one must, of course, prove that such stains are 
really due to blood by other methods, as this biologic test is merely for specific 
proteins and not for blood as such. These latter tests will be given in a later 
section. In this discussion the writer quotes largely from Uhlenhuth and 
Weidanz.^ 

The Anti-serum. 

This is the serum of the animal immunized against the proteins of the 
blood, exudates or secretions of a particular species of animal. These pro- 
teins are usually those of the human as this type is of especial importance in- 
medico-legal investigations. However, in the larger laboratories where 
many different serological tests are being frequently made, animals are kept 
on hand, which have been immunized against the proteins of most domestic 
animals, so that an absolute differentiation and identification of a stain may 
be made. 

The preparation of the anti-serum is as follows: A rabbit is injected intra- 
peritoneally with 5 to 10 c.c. of whole blood or serum. At intervals of 3 to 
5 days other similar injections are given until six or eight in all have been 
made. About a week after the last injection, a few drops of blood are 
drawn from a puncture in the ear of the animaFand are allowed to clot in a 
small test-tube. The potency of the seruni is then tested by permitting a few 
drops of the separated serum to fall into a small test-tube containing about 
I c.c. of a I to 1,000 dilution of dried homologous blood in physiological salt 
solution. In this preliminary potency test as in the later precipitin test it is 
important that the dilution of the solution under investigation should be 

^ See last chapter for a discussion of the phenomena of anaphylaxis brought about by 
such injections. 

2 SeeHektoen, Jour. Infect. Dis., 1914, XIV, 403; Bing and Trier, Ugesk. f. Laeger, 1914^ 
LXXVI, 2117; Bulger, Jour. Infect. Dis., 1916, XIX, 832; Smith, Jour. Med. Res., 1916,, 
XXXIV, 169; Bayne- Jones, Jour. Exper. Med., 191 7, XXV, 837; Stokes and Stoner, Bostl 
Med. and Surg. Jour., 1917, CLXXVII, 65; Hektoen, Jour. A. M. A., 1918, LXX, 1273;, 
Kominami and Kusakari, Kyoto Igaku Zasshi, 1918, XV, 4; Chem. Abs., 1919, XIII,, 
2920; Hektoen, Fantus and Portis, Jour. Infect. Dis., 1919, XXIV, 482, have applied this 
test to detection of occult blood in feces. 

2 Prakt, Anleitung z. Ausfiihrung des biol. Eiweissdifferenzierungsverfahrens, Jena, 
1909. See, also. Leers, Die forensische Bluntuntersuchung, Berlin, 1910. 



678 DIAGNOSTIC METHODS 

about I to 1,000. One may recognize this proper dilution by the fact that the 
solution forms a layer of foam on shaking and, further, on heating and adding 
to I CO. of the solution a drop of 25 per cent, nitric acid, only a slight 
opalescence obtains. If the addition of the anti-serum to the i to 1,000 
dilution of dried blood causes a turbidity in i to 2 minutes, the serum is 
sufficiently active for the test and the animal may be bled. 

The blood is now withdrawn, either in small amount as desired for im- 
mediate use or the animal may be completely bled. The blood is collected in 
wide-mouth test-tubes, which should be then plugged with cotton and placed 
in a slanting position until the blood is coagulated. The serum, which must 
be clear and sterile, is removed to small test-tubes, which are plugged with 
cotton, sealed with paraffin, and stored in a eool place. This is the anti-serum. 
It should be sufficiently potent to produce a turbidity almost at once, at 
latest in I to 2 minutes, in a i to 1,000 dilution of homologous blood in 0.9 
per cent. NaCl solution.^ 
Solution of the Blood or Stain. 

As a preliminary precaution, it is to be emphasized that all vessels and 
instruments used should be thoroughly clean and sterile and that all fluids be 
absolutely clear. 

If the blood or exudate be dried upon solid material, such as glass, knife 
blades, stone, wood, etc., it is carefully scratched off with a sterile instrument. 
This material is powdered and dissolved, as far as possible, in chemically pure 
physiologic salt solution contained in a sterile test-tube. If only traces of the 
stain are present, one may form a well of wax about the spots and place in this 
some of the salt solution. It is absolutely essential that no other solvent than 
0.9 per cent. NaCl solution be used, as otherwise erroneous reactions may 
occur or the usual precipitin reaction may not obtain. 

If the stain has penetrated clothing or other soft material, the spot is cut 
out with scissors, is finely divided, teased with a needle and placed in a small 
watch-glass or test-tube with a small amount of salt solution for one hour, or, 
if the stain be very old or has been exposed to marked changes in the weather, 
for 24 hours in a cool place. 

The fluid obtained in either of the above ways is filtered, first through 
hardened filter-paper (Schleicher and Schiill, Nr. 575, 603, or 605) and then 
through a small Berkefeld filter. This filtrate, which must be perfectly 
clear, should be neutralized to litmus either by the addition of sodium 
carbonate or tartaric acid solutions, as the case may be. Further, this 
filtrate should show distinct foam-formation on shaking. This is not always 
the case if the stain has been exposed to the sun and dust or has dried either 
upon fatty material or iron. Under these conditions, the precipitin reaction 
may not be seen in the usual time, but will obtain, as a rule, in five minutes. 

As previously stated, the dilution of this physiologic salt extract of the 

stain should be approximately i to 1,000. If sufficient stain can be scratched 

^ It is important to remember that such serum may contain a small amount of antigen 
(along with the precipitin) which may persist as long as 15 days after the last injection. 
See von Dungern, Centralbl. f. Bakteriol. ite Abt., Orig., 1903, XXXIV, 355; Uhlenhuth 
and Weidanz, Loc. cit., 221; Zinsser and Young, Jour. Exper., Med., 1913, XVII, 396. 



THE BLOOD 679 

from the surface of the material upon which it is found, this may be weighed 
and proper dilution made. Such procedures are usually impossible, so that 
we use the approximate method given on previous page. 

Technic of the Test. 

The reaction is carried out in the small test-tubes, which should be ap- 
proximately of the same size and thickness, should be thoroughly clean and 
sterile, should be numbered and arranged in a series of 10 tubes in a small rack. 

In tubes i and 2 are placed i c.c. of the above solution of the stain to be tested. 

In tube 3, I c.c. of a i to i,ooo dilution of known fresh human blood in 
physiologic salt solution. 

In tube 4, I c.c. of a i to i,ooo dilution of dried human blood in 0.9 per 
cent, salt solution. 

In tube 5, I c.c. of sterile physiologic salt solution. 

In tubes, 6, 7, 8, and 9, i c.c. each of a i to 1,000 dilution of fresh or dried 
blood of such domestic animals as may be easily obtained, as chicken, dog, 
horse and sheep blood. 

In tube 10, I c.c. of a physiologic salt extract of the material upon which 
the stain was found. 

To each of the above tubes, with exception of tube 2, is now added from a 
carefully graduated pipet o.i c.c. of the anti-serum, which has been tested for 
its potency as above described. To tube 2 is added o.i c.c. of normal clear 
rabbit serum. The anti-serum must be carefully added in such a way that it 
runs down the side of the tube and does not drop directly into the fluid. 
After this addition the tube must not be shaken. The reaction is carried out 
at room temperature. 

If the reaction be positive, a distinct clouding or turbidity will be observed 
immediately, or at latest within two minutes, in tubes i, 3, and 4 (all of which 
contain human blood), while the other tubes should remain perfectly clear. 
If the anti-serum has been properly added, the turbidity may be visible as a 
distinct contact ring which gradually increases upward. A negative reaction 
is shown by the lack of turbidity in any of the tubes. 

Specificity of the Reaction. 

Although this test is extremely delicate, yet it may happen that a stain, 
definitely identified as a blood-stain by other tests, may not react t)rpically to 
the precipitin test, owing to the small amount of protein matter in the salt 
extract. This is especially the case if the protein has been rendered insoluble 
by heat or destroyed by chemical agents or by putrefaction. Even in such 
cases a positive reaction may occasionally be obtained, Uhlenhuth and 
Beumer reporting positive results* after blood had been subjected to putre- 
faction for two years. 

As far as the influence of time upon this reaction is concerned, it may be 
said that blood-stains over 50 years old have been detected. The whole point, 
in this connection, is whether any trace of soluble protein remains. If so, the 
test will react positively, providing the dilution and the time of reaction be 
carefully regulated to suit the lessened concentration. 



It 



680 DIAGNOSTIC METHODS 

Heterologous precipitin reactions do not occur if the conditions of the test 
are properly maintained. If more concentrated blood solutions are used and 
the amount of anti-serum increased, such erroneous reactions may obtain. It 
is true that confusing reactions may arise in the differentiation of the blood of 
the closely related animals such, for instance, as the horse and donkey; goat, 
sheep and ox; dog and fox; hare and rabbit; chicken and pigeon; and especially 
man and the anthropoid apes (orang-outang, gorilla and chimpanzee). How- 
ever, the point of great medico-legal importance is that such close differentia- 
tions are rarely called for, as the question at issue is, usually, whether a given 
stain is or is not due to human blood. As the apes are relatively scarce in 
this country, confusion is seldom probable. This reaction may, therefore, be 
regarded as practically specific for the especial type of blood protein with 
which the immune serum reacts (all details of the test being properly regu- 
lated), only the proteins of the very closely related animals responding to the 
test in a way which might leave the slightest room for doubt as to the origin 
of a stain or of a protein. 

The substance in which the stain is found may, often, exert some retarding 
action on the test. Stains upon cloth or paper usually show prompt results, 
although those upon wall-paper may, at times, react atypically owing to the 
influence of the chemical of the dyes. Strong alkalies or acids prevent the 
reaction, but these are thrown out by neutralization before the test is applied. 
Tannic acid, derived from stains upon leather may prevent the reaction, al- 
though this is not always the case. Iron rust may retard but does not pre- 
vent the reaction. If the stain be upon soft clay, mortar, lime or fresh plaster 
so that an intimate mixture has occurred, the reaction will probably not obtain, 
according to Graham-Smith and Sanger, but if the stain be upon hard well- 
dried mortar no influence is seen, as demonstrated by Biondi. 

As previously stated, this active immune serum reacts promptly with the 
protein elements of exudates, sputum, pus, urine, feces, nasal and bronchial 
secretions, vaginal, lochial and seminal fluids, etc., so that a positive precipitin 
reaction means merely that a protein is present, which has the same origin as 
that against which the animal furnishing the anti-serum was immunized.^ 
Whether or not this is a blood protein is, in no way, a part of the test. This 
must be determined by other tests. 

C. Complement Fixation Test. 

As previously stated, the serum of an animal, which has been injected with 
bacteria or with washed blood corpuscles of an animal of a different species, 
acquires the property of dissolving homologous cells through the development 
of antibodies or immune bodies, which are specific for the cells injected. 
This process is known as bacteriolysis or hemolysis. Such sera are known as 
lytic; bacteriolytic, if the action is directed toward bacteria, hemolytic, if 

^ Robertson and his pupils have contributed some interesting articles on the blood rela- 
tionship of animals as displayed in the composition of the serum proteins. See Robertson, 
Jour. Biol. Chem., 191 2, XIII, 325; Woolsey, Ibid., 1913, XIV, 433; Thompson, Ibid., 
1915, XX, i; Briggs, Ibid., 7; Sutherland (Indian Jour. Med. Res., 1915, III, 205) reports 
a study of 6566 articles examined for blood stains. 



THE BLOOD 68 1 

toward blood cells. It is to be remembered, in this connection, that fresh 
blood serum of many species is hemolytic for the red cells of some, but not all, 
other species, the degree being relatively small. Moreover, we find that the 
blood of the same species may be markedly hemolytic for the homologous 
cells, as evidenced by the effects occasionally observed after transfusion. It 
is, therefore, evident that we may have in any serum both the immune and the 
natural hemolytic agents, the former being much the more effective. 

It has been shown that hemolysis, by means of an immune serum, may be 
prevented by heating the serum to 55 to s6°C. for one-half hour, the process 
being known as inactivation. Addition of a small amount of a fresh, isologous 
serum will restore (reactivate) the hemolytic activity. Hence, two separate 
factors must be concerned in hemolysis, the one a thermostable (heat-re- 
sisting) substance, the other a thermolabile (destroyed by heat) product. 
The first of these is called the amboceptor, the latter, the complement. 
Amboceptor is usually still active in serum which has been kept for a long 
period, while complement disappears from the serum in a short time even 
though it be kept on ice. The function of the amboceptor is to sensitize the 
cells, while that of the complement is to dissolve the cells so sensitized. 
Hemolysis is impossible without the combined action of both factors, either 
one singly having no effect. Complement can combine with the cell only 
through the medium of amboceptor, while this union does not take place 
unless amboceptor and antigen (the blood cells) are homologous. In other 
words, the cells can be hemolyzed only by homologous serum in the presence 
of complement. It is evident, on the other hand, that hemolysis must occur 
when the proper hemolytic trinity is present. The amboceptor and com- 
plement bear definite quantitative relations to one another in this process, an 
increase of one factor permitting the use of a less amount of the other. 

Complement may combine itself equally well with bacteriolytic or hemo- 
lytic amboceptor providing the homologous antigen be present. If, there- 
fore, we mix bacterial emulsions with the corresponding inactivated immune 
serum and complement, this latter must attach itself (become fixed) to the 
bacterial cell through the medium of the amboceptor, so that the later addi- 
tion of hemolytic amboceptor and washed cells will not result in hemolysis, 
owing to the fact that the complement originally present has been fixed to 
such an extent that none is left to combine with the hemolytic amboceptor. 
This phenomenon, discovered by Bordet and Gengou, is known as "fixation 
of complement" or as "deviation of complement." It is decidedly a quan- 
titative as well as a qualitative reaction. Had complement remained free 
or been added in excess of the amount necessary to act upon the bacterial cell, 
a certain amount of visible hemolysis must have occurred, and might, even, 
have been complete, had sufficient hemolytic amboceptor been present. 
These quantitative elements are the most important ones in the application 
of the test to be discussed later. 

This test has been used for diagnostic purposes in many infectious diseases; 
for instance,, in typhoid fever by Widal and Lesourd as well as by Hirschfeld, 
in cerebrospinal meningitis by Cohen, in whooping cough by Bordet and Gen- 



682 



DIAGNOSTIC METHODS 



I i 



gou, in scarlet fever by Besredka and Dopter and Foix and Mallein, in sys- 
temic gonococcic infections by Miiller and Oppenheim, Bruck, Albarran and 
Jungano. Its most important and frequent application is in the sero-diag- 
nosis of syphilis, as advanced by Wassermann, Neisser and Bruck and, inde- 
pendently, by Detre. 

It is evident that the application of this complement fixation test leads, 
just as in the case of the agglutination and precipitin tests, to the identifica- 
tion of an antibody, provided one has the homologous antigen, or vice versa, 
the antigen and antibody necessarily being homologous and, hence, specific, 
the one for the other. It had been shown by Klebs, Metchnikoff and Roux 
that syphilis could be transmitted to monkeys by inoculation with human 
syphilitic material (antigen) . Wassermann and his coworkers then found that 
the serum of these syphilitic apes contained an antibody not found in normal 
serum. Having this antibody, it was an easy step to show its relation to 
syphilitic antigen and to apply the complement fixation test in the detection 
of antibodies in the serum of patients infected with syphilis.^ As the culti- 
vation of the treponema pallidum has only recently been achieved by Noguchi, 
no direct method of detecting these antibodies is available. ^ As indirect 
methods must be used, the antigen employed in the early studies was an ex- 
tract of syphilitic tissues in the active stage of the disease, as these contain 
large numbers of the causative spirochete. Later workers have shown that 
this antigen need not be specific, as alcoholic extracts of certain normal or- 
gans, crude tissue lecithin, or, even, salts such as sodium glycocholate or 
oleate, may serve quite as well in the reaction. Although this fact lessens 
the true specificity of the test, from the biologic standpoint, there is no doubt 
of the clinical value of the reaction. The so-called syphilitic antigen must be 
either a normal substance or one possibly associated with an unknown prod- 
uct which actually produces a specific result. However this may be, it is a 
well established fact that, if the serum of a patient infected with syphilis be 
treated with antigen and complement, no hemolysis occurs on the later addi- 
tion of hemolytic amboceptor and red cells because the complement is bound 
to the antigen through the medium of the homologous amboceptor; while if 
the patient is non-syphilitic marked hemolysis occurs as complement is free 
to act with the hemolytic system. This is the basis of the Complement Fixa- 
tion Test as applied to syphilis by Wassermann and his colleagues. Although 
the antigen need not be specific, yet the test itself is remarkably specific being 
given by only a few non-syphilitic conditions as will be seen later. 

In the actual technic of the test, many modifications have been advised, 
the variations consisting either in the antigen or in the cells to be hemolyzed 
or in both. Five essential factors enter into the reaction whatever be the 
modification employed. These are (i) syphilitic antigen, (2) syphilitic anti- 
body, (3) erythrocytes (antigen), (4) hemolytic amboceptor and (5) com- 
plement. These factors must be accurately adjusted to one another, as 
serious errors may arise if the quantitative relations are not observed. The 

^ See Krauss, Biochem. Ztschr., 1914, LXIV, 222. 
2 See foot-note on page 622 



M 



i 



THE BLOOD 683 

writer will describe only the original Wassermann and the Noguchi methods, 
as these are, in his opinion, the most reliable ones to follow.^ 

a. The Wassermann Reaction. 
Preparation of Antigen. 

Since the discovery of the fact that the antigen used in this test was not 
absolutely specific, in the biologic sense, for the syphilitic antibodies, many 
different antigens have been advocated. The writer selects those which have 
promised the best results, although he believes that Noguchi's antigen and, 
especially, Sachs' alcoholic heart antigen with cholesterin are by far the most 
reliable and constant ones introduced. 

a. Aqueous Extracts. 
I. Wassermann's Method. 2 

The liver or spleen of a syphilitic fetus is ground up in a mortar with sand 
or, preferably, minced in a meat grinder. The hash is placed in a dark-colored 
flask and mixed with 0.9 per cent, sodium chlorid solution containing 0.5 per 
cent, phenol. The proportion is i gram of substance to 4 c.c. of the salt solu- 
tion. This mixture is shaken for 24 hours in a shaking machine and is then 
allowed to settle or is centrifuged. The reddish-brown opalescent superna- 
tant liquid, which is the antigen, is decanted into dark bottles, tightly stop- 
pered and kept on ice. On standing, a precipitate forms, which must not be 
disturbed by shaking. This aqueous antigen appears to be rather unstable 
and shows, sooner or later, some inhibition (anticomplementary action) with 
normal serum, although Citron^ finds such antigens active even after 11 
months. Before use the activity of this extract, as well as of all others, must 
be quantitatively determined by the method later outlined. The usual doses 
are 0.2 (maximum) and o.i (minimum) c.c. As a general rule, we use only 
such an amount of antigen, the double dose of which shows no binding of 
complement. 

h. Alcoholic Extracts. 
I. Method of Porges and Meier. ^ 

The liver of a syphilitic or normal fetus is hashed and extracted for 24 
hours with five times its weight of absolute alcohol. Filter through filter- 
paper and distill the alcohol from the filtrate in vacuum at 4o°C. A syrupy 
residue remains which is suspended in physiologic salt solution containing 0.5 
per cent, phenol, using 100 parts of salt solution to i of syrup. Filter through 
filter-paper. The usual dose of this antigen is 0.2 to 0.3 c.c, although less 
quantities may be used as determined by methods of standardization. 

1 Hecht (Wien. klin. Wchnschr., 1909, XXII, 265) and later Weinberg (Ann. de I'lnst. 
Pasteur, 1912, XXVI, 427) utilize the natural amboceptor and complement present in 
unheated human serum. See, also, Gradwohl, Jour. Am. Med. Assn., 19 14, LXIII, 240; 
Kolmer, Jour. Immunol., 1916, II, 23; Gradwohl, Jour. A. M. A., 191 7, LXVIII, 514; 
Am. Jour. Syph., 1917, I, 450; Williamson, Jour. Lab. and Clin. Med., 1917, II, 658; 
Christian, Ibid., 1919, III, 613. 

2 Deutsch. med. Wchnschr., 1906, XXXII, 745. 

3 Die Technik der Bordet-Gengouschen Komplementbindungsmethode. in Handbuch of 
Kraus and Levaditi, Jena, 1909, p. 1076; Die Methoden der Immunodiagnostik und Im- 
munotherapie, Leipzig, 1910. See, also, Bruck, Die Serodiagnose der Syphilis, Berlin, 1909. 

* Berl. klin. Wchnschr., 1908, XLV, 731. 



684 DIAGNOSTIC METHODS 

Instead of this antigen, these workers use, also, a i per cent, solution of 
commercial lecithin in phenol-containing physiologic salt solution. This 
solution is stable if kept in the ice box. It has the disadvantage that the 
smaller doses may inhibit hemolysis, while the larger ones may, themselves, 
cause hemolysis. The stronger luetic sera require a larger dose (o.i c.c), 
while the weaker sera use less (o.oi c.c.) of lecithin. Hence we employ an 
average dose of 0.05 c.c, which may be compensated by the use of four to six 
times the titrated dose of amboceptor and complement. 

2. Method of Landsteiner, Miiller and Poetzl.^ 

The muscular tissue, free from fat, of a guinea-pig heart is rubbed up in a 
mortar and extracted with 95 per cent, alcohol, using 50 c.c. for every gram of 
substance. Heat for several hours at 60° C. and filter through filter-paper. 
The filtrate is stable at room temperature for long periods. It is used in doses 
of 0.3 to 0.05 c.c. 

3. Method of Sachs. 2 

As it had been shown^ that the addition of cholesterin to crude alcoholic 
extracts furnished an antigen of especially powerful and specific properties, 
although cholesterin itself had no antigenic effect, Sachs advocated a choles- 
terin-fortified extract of beef heart or liver or of guinea-pig heart. Mcintosh 
and Fildes^ find that human heart furnishes a superior antigen. This latter 
material is used in the writer's laboratory, the antigen being prepared as 
follows : 

The muscular tissue of a human heart, normal or pathologic, is freed from 
gross fat, finely minced, weighed, and ground up in a mortar with absolute 
alcohol in the proportion of i gram of tissue to 10 c.c. of absolute alcohol. 
Transfer the mixture to a stoppered bottle and shake for one and one-half 
hours at room temperature. Filter through paper and preserve in an amber 
stoppered bottle. Although the activity of this extract does not change for 
2 or 3 months, a more reliable preparation may be had by preparing it fresh 
each month. To 5 parts of this 10 per cent, alcoholic extract of human heart 
add 4 parts of a i per cent, solution of cholesterin in absolute alcohol. This 
forms the stock antigen. When used in the test a fresh i to 10 emulsion in 
normal saline is prepared by adding the stock antigen drop by drop with con- 

1 Wien. klin. Wchsnchr., 1907, XX, 1565; 1908, XXI, 282; Ruediger (Jour. Infect. Dis. 
1 9 19, XXIV, 31 and 204) has shown that alcoholic extract of human heart, beef heart or 
rabbit heart give more reliable results than those extracts prepared from guinea pig heart. 

2 Berl. klin. Wchnschr., 191 1, XLVIII, 2066. 

^ See Browning, Cruickshank and McKenzie, Biochem. Ztschr., 1910, XXV, 85; Jour. 
Path, and BacterioL, 1910, XIV, 484; Browning and McKenzie, Diagnosis and Treatment 
of Syphilis, Philadelphia, 191 2; Desmouliere, Presse Med., 1913, XXI, 898; Kolmer, 
Laubaugh, Casselman and Williams, Arch. Int. Med., 1913, XII, 660; Thomas and Ivy, 
Jour. Am. Med. Assn., 1914, LXII, 363; Orkin, Berl. klin. Wchnschr., 1914, LI, 690; 
Thompson, Jour. Am. Med. Assn., 1914, LXII, 1458; Field, Ibid., 1620; Arch. Int. Med., 
1914, XIII, 790; Judd, Jour. Am. Med. Assn., 1914, LXIII, 313; Owen and Snure, Jour. 
Mich. Med. Soc, 1914, XIII, 422; Interstate Med. Jour., 1914, XXI, 1281; Weston, Jour. 
Med. Research, 191 4, XXX, 377; McClure, Ibid., 455; Walker, Arch. Int. Med., 1914, 
XIV, 563; Hopkins and Zimmermann, Am. Jour. Med. Sc, 1914, CXLVIII, 390; Kolmer 
and Schamberg, Ibid., 1915, CXLIX, 365; Cornwall, New York Med. Jour., 1915, CI, 844; 
Henes, Jour. Am. Med. Assn., 191 5, LXIV, 1969; McClure and Lott, Am. Jour. Med. 
Sc, 1916, CLI, 712. 

^ Ztschr. f. Chemotherap., Orig., 191 2, I, 79. 



THE BLOOD 685 

stant stirring to normal saline in proportion of i part of former to 9 of latter. 
Walker and Swift^ believe that syphilitic patients under treatment should be 
treated until negative reactions obtain with a i to 6 dilution of this extract, 
as this gives a much more delicate reaction. The point to be remembered is 
that not more than one-fourth of the anti-complementary dose of this antigen 
should be used, although it is possible to use one-third of this dose providing 
very slight degrees of inhibition are disregarded and such tests called negative. 
This antigen has the great advantage that its antigenic and anti-comple- 
mentary properties are practically the same, although prepared from different 
hearts. The only criticism of this antigen is that it may yield non-specific 
fixation tests in a certain number of cases, but its greater sensitiveness enables 
it to catch a certain number of cases that are doubtful with other antigens. 
The tests run through uniformly and always show the same degree of 
activity, making the interpretation of the results a matter of greater accu- 
racy. This antigen may be recommended as a standard one and should be 
used in every test made, even though any number of antigens be used with it. 

c. Acetone -insoluble Extracts. 

As it has been shown that the alcoholic extracts of various organs contain 
variable amounts of "syphilitic antigen," Noguchi^ advocates the use of the 
alcohol and ether soluble but acetone insoluble extract of organs. This frac- 
tion contains lecithin and other phosphatids. 

Method of Noguchi. 

Extract a mashed paste of liver, heart, or kidney of man, ox, guinea-pig, 
rabbit, or dog with 10 parts of absolute alcohol at 37°C. for several days. 
Filter through filter-paper and collect the filtrate. The latter is then brought 
to dryness by evaporation with the aid of an electric fan or in a vacuum at 
4o°C. Take up the dried residue with ether and allow the turbid solution to 
stand in a covered dish over night in a cool place. Decant the clear ethereal 
solution into a clean beaker and concentrate by evaporating the most of the 
ether. Mix this concentrated ethereal solution with 10 volumes of pure 
acetone. Allow the precipitate to settle and decant off the supernatant fluid. 
This antigen is a light brownish precipitate which gradually becomes sticky on 
exposure to the air. This product usually has no hemolytic action upon 
human red cells, but may occasionally show this property. Further, it may, 
also, show an anticomplementary action. Three-tenths gram of this acetone- 
insoluble fraction is dissolved in i c.c. of ether and is mixed with 9 c.c. of 
methyl alcohol. If any precipitate forms or is left undissolved, remove it by 
centrifugation. This stock solution remains unaltered for a long period. 
For the actual test an aqueous emulsion is prepared by mixing i c.c. of the 
stock solution with 9 c.c. of physiologic salt solution. This produces a clear 
opalescent solution containing 0.3 per cent, of the original lipoids. One- 

^ Jour. Exper. Med., 1913, XVIII, 75. 

2 Jour. Exper. Med., 1909, XI, 84; Serum Diagnosis of Syphilis, Philadelphia, 191 2. See, 
also, L'Esperance and Coca, Jour. Immunol., 1916, I, 129; in the new hemohemolytic sys- 
tem test of Noguchi, this antigen is the only one that may be reliably employed. 



686 DIAGNOSTIC METHODS 

tenth c.c. of this emulsion is used in the test later described. This emulsion 
is kept on ice as it is unstable in this form.^ 

Antigen Paper. — Instead of keeping a stock solution of antigen as above 
described, Noguchi has introduced the use of paper impregnated with antigen 
for the test. These papers are especially serviceable to the general worker 
v/ho may wish to employ this test but they do not hold their properties longer 
than three months. These are far inferior to the liquid antigen but may be 
used in an emergency. Weigh out about 1.2 grams of the sticky extract and 
dissolve in about 20 c.c. of ether. Have ten sheets of filter-paper, 10 by 10 
cm. in dimensions, laid one upon the other in a clean glass dish. Pour over 
these the lipoid solution and saturate the paper evenly. Separate each sheet 
as quickly as possible and lay flat on a clean sheet of unbleached muslin. 
Within 10 minutes the paper is ready for use. Before assigning the dimen- 
sions for each tube in the fixation test, the antigen paper should be titrated. 
This is done as follows: Cut the paper into equal width, say 5 mm., and use 
increasing lengths of this strip for standardization, starting with i mm., 2 
mm., etc. The principle of this standardization will be discussed later. The 
strips may be marked in sections, each representing the required dimensions, 
and put into sealed tubes for preservation. 

The Syphilitic Antibody. 

The immune serum may be withdrawn from the patient by the method 
given under blood cultures. The blood, thus obtained, is allowed to coagu- 
late in a large test-tube. The serum is then drawn off with a pipet into small 
test-tubes and inactivated by heating to 56°C. for one-half hour. 

In the writer's laboratory, the custom is to withdraw the blood from the 
median basilic vein by means of a pipet of about 20 c.c. capacity. ^ One end of 
this pipet is attached to a rubber tube with glass mouthpiece, while the other 
end bears a steel or platinum-iridium needle attached with rubber tubing. 
Five to ten c.c. of blood are withdrawn and placed immediately in a small test- 
tube where it is allowed to clot. After about 20 minutes, the clot is separated 
from the walls of the tube and the tube is then centrifuged to obtain the clear 
serum. If the specimen is drawn by the physician and is to be forwarded to 
the laboratory, it is essential that the cells be separated from the serum before 
the specimen can be sent. ^ Such specimens give reliable results, but if the cells 
are allowed to remain in contact with the serum for any length of time, so 
much hemolysis occurs that the test can not be accurately made by a distant 

^ Noguchi has recently (Jour. Am. Med. Assn., 191 2, LVHI, 1163) prepared a specific 
antigen from cultures of the treponema pallidum. The most interesting finding, based upon 
comparative studies with this antigen and with the lipoid antigens, is that there is no indica- 
tion, in cases of active syphilitic orchitis, of a sufficient amount of specific antibodies to bind 
complement with the pallida antigen, although a strong positive Wassermann reaction 
obtains. See Craig and Nichols, Jour. Exper. Med., 191 2, XVI, 336; Kolmer, Williams and 
Laubaugh, Jour. Med. Research, 1913, XXVIII, 345; Varney and Baeslack, Jour. Am. Med. 
Assn., 1913, LXI, 754. 

2 See Parvu, Arch, des Mai. du Coeur, 191 5, VIII, 439. 

^ Ruediger (Phil. Jour, of Sc, 191 7, XI, i and 87) states that glycerin keeps the serum 
sterile and does not noticeably influence the reaction. Hence this may be used in cases 
where it is necessary to delay the test for some time. 



THE BLOOD 687 

laboratory. The serum is inactivated by heating to 56°C. for one-half hour 
either immediately after centrifugation or on receipt of the specimen. 

This inactivation is absolutely essential provided aqueous or alcoholic 
extracts are used as antigens, because these antigens contain various proteins, 
which are liable to give non-specific proteotropic fixation with active sera. 
Noguchi has shown that this property is lost if the serum be inactivated. 
Moreover, with his antigen (acetone-insoluble lipoids) no such proteotropic 
fixation occurs, so that active sera may be employed in his test, although the 
inactivated serum works quite as well. 

Suspension of Red Cells. 

In the Wassermann test, the corpuscles to be hemolyzed are those of the 
sheep. The blood may be obtained from an abbatoir or sheep may be kept in 
the animal rooms of the laboratory and bled from the ear as desired. The 
blood is received directly in a more than equal amount of i per cent, sodium 
citrate solution in physiologic salt solution or it maybe defibrinated by shaking 
with glass beads or by whipping with wires or bristles. It is then centrifuged 
and the supernatant fluid drawn off. The cells are thoroughly mixed with 
physiologic salt solution, again centrifuged and the clear fluid withdrawn. 
This process is repeated two or three times until the corpuscles are free from 
serum. The sedimented corpuscles are now drawn up with a graduated pipet 
and added to 19 volumes of salt solution. A 5 per cent, suspension is thus 
obtained, of which i c.c. is used in the test. If the centrifuge tube be cali- 
brated, one may use this directly in making the suspension. Such suspen- 
sions may be used for two or three days, especially if kept in the ice box, but 
the}^ should be discarded if any trace of hemolysis appears in the tube.^ 

Hemolytic Amboceptor. 

The hemolytic serum is obtained from rabbits which have been injected 
with washed sheep corpuscles. If the blood is received from the abbatoir, it 
is usually contaminated to such an extent that it must be sterilized by heating 
to 6o°C. for one-half hour. The cells are obtained as above described and 
washed very thoroughly with salt solution. When free from serum, their 
volume is made up to that of the original defibrinated blood with physiologic 
salt solution. The immunization is best carried out by injecting a rabbit, 
intraperitoneally, with 2, 4, 8, and 12 c.c. of cells at intervals of 4 or 5 days. 
Ten days after the last injection a few c.c. of blood are obtained from an ear 
vein of the rabbit and the serum tested for its hemolytic effect upon sheep 
corpuscles. If it produces hemolysis in dilutions of i to 600 or more, the 
animal is bled from the carotid artery, the blood being collected in a series of 
sterile test-tubes. These are placed in the ice box, the blood allowed to coagu- 
late and the serum is drawn off. This serum is then inactivated and put 
into small sterile tubes, which should be sealed and kept on ice. This ma- 
terial keeps well, but its strength must be determined at frequent intervals. 

^ See Trossarello (Rif. Med., 1914, XXX, 95) also, Rous and Turner (Proc. Soc. Exper. 
Biol, and Med., 1915, XII, 122, Jour. Exper. Med., 1916, XXIII, 219 and 239) for methods 
of preserving these blood ceUs. Reimann, Jour. Lab. and Clin. Med., 1917, II, 200; 
Dreyer and Gardner, Lancet, 1919, II, 687. 



688 DIAGNOSTIC METHODS 

It is wise to inject several animals at the same time, so that one may surely 
yield a satisfactory serum of high hemolytic power/ 

Complement. 

Normal guinea-pig serum is used as complement in all modifications of this 
test. 2 The animal is anesthetized and the blood drawn from the carotid, as 
above described, into sterile test-tubes in which it is allowed to coagulate. 
The serum is then drawn off with a sterile pipet, placed in small sterile test- 
tubes and kept on ice. 

As complement deteriorates rapidly (being of little use after 48 hours) the 
blood is, preferably, withdrawn by aspiration directly from the heart.^ The 
animal will usually survive the loss of 5 to 10 c.c. of blood, providing the heart 
has not been badly lacerated by repeated punctures. After two weeks the 
animal may be used for a fresh supply of complement. It is wise to have quite 
a number of these animals on hand so that fresh material may constantly be 
available. It is absolutely unnecessary to sacrifice an animal each time a 
few tests are to be made. When used complement is diluted i to 10 with 
physiologic salt solution.^ 

Standardization of Reagenfs. 

This standardization must always be preliminary to the actual perform- 
ance of the test. Everything depends upon the proper adjustment of the 
various factors entering into the test, so that especial care must be taken to 
find out just what quantities of each reagent to use. Without such precau- 
tions, little, if any, value can be attached to any report. 

Titration of Hemolytic Amboceptor. 

Prepare a series of dilutions of the inactivated serum beginning with i to 
300 and extending to i to 3,000. This may be readily accomplished by dilu- 
ting 0.1 c.c. of serum to 30 c.c. with physiologic salt solution and using this 
I to 300 dilution as the basis of further dilutions. Arrange a series of small 
test-tubes and place in each 0.25 c.c. of these diluted sera, beginning with the 
highest concentration and extending to the lowest. Now add 0.25 c.c. of a 
I to 10 dilution of complement and 0.25 c.c. of a 5 per cent, suspension of 
sheep corpuscles to each tube. Bring the total volume up to 1.25 c.c. with 
physiologic salt solution. All tubes are placed in the incubator at 37°C. for 

1 See Coca, Jour. Infect. Dis., 1915, XVII, 361; also, Morgenroth and Bieling, Biochem. 
Ztschr., 1915, LXVIII, 85. 

2 Noguchi (Jour. Exper. Med., 1918, XXVIII, 43) has introduced a new modification 
of his test using a homohemolytic system by the employment of human complement. 
See, also, Lewis and Newcomer, Ibid., 1919, XXIX, 351; Bruynoghe, Bull. acad. roy. med. 
Belg., 1919, XXIX, 209, 222, and 231. 

•'* See Austin (Jour. Am. Med. Assn., i9i4,LXII, 868) and Weston (Jour. Med. Research, 

191 5, XXXII, 391) for methods of preserving complement; Thompson, Jour. A. M. A., 

1916, LXVI, 652; Rhamy (Jour. A. M. A., 1917, LXIX, 973) advises the use of sodium 
acetate as a preservative, mixing 4 parts of complement with 6 parts of a 10 per cent, 
solution of sodium acetate in 0.9 per cent, sodium chloride solution. Emerson (Jour. 
Lab. and Clin. Med., 1919, V, 62) advocates the use of chloroform. 

^See Tissot (Compt. rend. Acad, de Sc, 1914, CLVIII, 1525); Thorsch (Biochem. 
Ztschr., 191 5, LXVIII, 67); Ottenberg, Reuben and Frazier (Jour. Infect. Dis., 1915, XVI, 
119) for studies of the nature of complement. Cole (Cleveland Med. Jour., 1915, XIV, 273) 
show the presence of anti-sheep amboceptor in guinea-pig's blood — Mliller, Wien. klin. 
Wchnschr., 1916, XXIX, 1239; Ruediger, Jour. Infect. Dis., 1919, XXIV, 120. 



THE BLOOD 689 

two hours and are then allowed to stand over night in the ice box. The 
highest dilution (smallest amount of amboceptor) in which complete solution 
of the cells occurs is taken as the strength of the serum. This is called one 
amboceptor unit. In performing the Wassermann test, two units of ambo- 
ceptor are used. The titer of the amboceptor remains fairly constant, but 
it is wise invariably to determine its value when fresh complement is to be 
used, owing to the marked influence of these factors on one another. 

Instead of using the above method of standardization, one may resort to 
the drop method of Landsteiner, Miiller and Poetzl, which simplifies the tech- 
nic but, in so doing, impairs the accuracy of the result, in the writer's opinion, 
as the drops from different capillary or other pipets are seldom of the same 
size. By this method one determines what dilution of amboceptor in i drop 
dose will hemolyze in one-half hour, in an incubator, i drop of 50 per cent, 
suspension of sheep's corpuscles with i drop of complement, 10 drops of 
salt solution being added as diluent. Twice the strength of the titer is used 
in the test. 

Titration of Complement. 

Having obtained the value of the amboceptor unit, one may use decreasing 
amounts of complement with one unit of amboceptor in the same way as in 
the above titration, the dilution of the complement running from i to 2 to i to 
20. Complement is usually used in a dilution of i to 10, i c.c. being em- 
ployed in the test. This is, however, open to objections as it is not absolutely 
quantitative. Noguchi has pointed out the fact that variation in the amount 
of complement used influences the amount of amboceptor. " If less than one 
unit of amboceptor is used hem^olysis will always be incomplete, even with 
more than one unit of complement. Likewise, if with one amboceptor unit 
there is combined less than one unit of complement, hemolysis can not be 
complete. If with more then one unit of amboceptor there be used less than 
one unit of complement, hemolysis may be complete or incomplete according 
to the relative amounts of each factor used.^ In the presence of many units 
of amboceptor hemolysis may be complete when but a small fraction of the 
complement unit is present." 

Titration of Antigen. 

The degree of dilution in which the antigen is to be used must be deter- 
mined for each extract by testing it both against normal and syphilitic sera. 
Its concentration must be such that it does not prevent hemolysis with normal 
sera, while it will promptly inhibit hemolysis in presence of complement with 
syphilitic sera.^ 

Set up two parallel series of tubes. Into each tube of one series place 0.25 
c.c. of a I to 5 dilution of known syphilitic serum and into each tube of the 
second series place 0.25 c.c. of a i to 5 dilution of normal serum. Dilutions of 
the antigen are now made, w^ith physiologic salt solution as the diluent, begin- 

^ See Kromayer and Trinchese, Med. Klin., 191 2, VIII, 404 and 1670, who use an abso- 
lute minimum of complement in their "refined" Wassermann test; also, Thompson, Arch. 
Int. Med., 1914, XIII, 904. 

2 See Ottenberg, Jour. Immunol., 1916, II, 47; Denzer, Jour. Infect. Dis., 1916, XVIII, 
631. 

44 



690 DIAGNOSTIC METHODS 

ning with i to i and running up to i to 20 or higher if necessary. Into the 
first tubes of the above series place 0.25 ex. of the first antigen dilution, into 
the second tubes of each series place the second dilutions and so on until all 
are accounted for. To all of the above tubes add 0.25 c.c. of complement 
(i to 10) and incubate at 37°C. for one hour. Now add 0.25 c.c. of hemolytic 
amboceptor in twice the maximum dilution found to cause complete hemo- 
lysis (two units) and 0.25 c.c. of a 5 per cent, suspension of sheep corpuscles. 
Place in an incubator for two hours and then in ice box over night. No hemo- 
lysis should be observed in the tubes containing syphilitic serum except in 
those containing the higher dilutions, while in the tubes with normal serum 
hemolysis will occur in all tubes except those with the lower dilutions of 
antigen. Hemolysis in the tubes with S3^hilitic sera is due to the normal 
hemolytic power of the antigen, while the lack of hemolysis in the tubes with 
normal sera is due to the anticomplementary action of the antigen. In 
carrying out the Wassermann test, antigen is used in one-half the lowest dilu- 
tion (largest amount of antigen) which gives complete hemolysis with normal 
sera, this being taken as one antigen unit. Thus if a dilution of i to 5 gave 
complete hemolysis, a dilution of i to 10 would be used in the actual test. 

If one uses the drop method, as mentioned above, i drop of antigen (undi- 
luted) and I drop of complement (undiluted) are mixed with 10 drops of salt 
solution. The mixture is incubated one-half hour and i drop of amboceptor 
and I drop of a 50 per cent, suspension of corpuscles are added. Hemolysis 
should occur within one-half hour. If it does not, anticomplementary action 
is marked and less antigen must be employed in the test. 

Having determined the strength of the antigen, a portion of the stock solu- 
tion is diluted with salt solution so that i c.c. equals one unit. In testing the 
antigenic properties of this antigen, one may use it with a large number of posi- 
tive and normal controls, or, preferably, run it in parallel series with an antigen 
of known value. If the results agree, it may then be substituted for the old 
antigen. The titer being known, the antigen may be used for subsequent 
tests without redetermination of this value. 

Technic of the Test. 
** Original Wassermann Method. 

All of the glassware used in the test must be kept thoroughly clean and 
dry, but need not be sterile. The pipets, which should be both graduated 
to o.oi and o.i c.c, should, preferably, be used with only one reagent. If 
they are employed with others, they must be thoroughly rinsed in salt solu- 
tion between such applications. After using, wash them well in salt solu- 
tion (never with soap and water), allow them to stand in a tall cylinder of 
distilled water for a few hours and dry them in an oven. The test-tubes used 
in this test are about 14 cm. in length by 1.5 cm. in diameter, while the some- 
what smaller ones (i by 10 cm.) may be employed in the Noguchi modification. 

Before actually performing the test, the reagents are prepared and stand- 
ardized as previously described or the amboceptor may be titrated at the begin- 
ning of the test, as it is not added until the preliminary incubation of the serum 



THE BLOOD 69 1 

with the antigen and complement. The sera of the patient and of the 
known positive and normal controls must all be inactivated by heating to 
56°C. for one-half hour.^ The complement is diluted i to 10 with physio- 
logic salt solution and the suspension of washed sheep corpuscles prepared. 
When these preliminaries are attended to one may proceed with the test 
itself. 

Arrange a double row of test-tubes, the front tube of each set to receive 
the antigen and the back row the control tube without antigen. One set of 
tubes must be placed for the unknown serum, one set for the positive and one 
set for the normal control; while other controls are arranged as spoken of later. 

1. Into both tubes of each set place 0.2 c.c. of the serum to be tested. 

2. To all tubes, add i c.c. of complement (diluted i to 10). 

3. To tubes in front row only, add 0.2 c.c. of antigen (aqueous extract of 
syphilitic liver) . 

Make up volume of each tube to 3 c.c. with salt solution, mix contents by 
shaking and incubate at 37°C. for one hour.^ 

4. Add to each tube i c.c. of antisheep amboceptor (two units). 

5. Add to each tube i c.c. of 5 per cent, suspension of sheep corpuscles. 
Mix the contents of the tube thoroughly, incubate at 37°C. for two hours 

and place the tubes in the ice box over night. At the end of this period the 
results are read. 

Controls. 

1. Positive syphilitic serum. To be run as above. 

2. Known negative serum. To be run as above. 

3. A tube containing complement and two units of antigen, to prove that 
no anticomplementary action of the antigen obtains. 

4. A tube containing complement, to show that the hemolytic system is 
active. 

5 . A tube containing antigen alone, to show absence of lytic power of antigen. 
All controls are brought up to 3 c.c, incubated and, then, amboceptor and 

sheep corpuscles are added as above. 

If the test is properly conducted, hemolysis will occur in every control tube 
excepting in the tube containing syphilitic serum and syphilitic antigen (the 
positive control) and in the tube of control 5. In the series of unknown sera, 

^ Gramenitzki (Biochem. Ztschr-., 191 2, XXXVIII, 501) and Fenyvessy (Ibid., 191 2, XL, 
353) have shown that if the serum be not heated to complete inactivation, the complement 
is restored on allowing to stand for some time. 

^ Recent work has shown that a lower temperature of the first incubation yields a higher 
percentage of positive results in known positive cases and, also, that this method, catches 
more of the less easily recognized cases than does the usual incubation at 37°C. Smith 
and MacNeal (Jour. Immunol., 191 6, II, 75; Jour. Infect. Dis., 191 7, XXI, 233) advocate 
incubation in the refrigerator at approximately 8°C. for 4 to 24 hours. Dean (Jour. 
Path, and Bact., 1917, XXI, 193) and Ruediger (Jour. Infect. Dis., 1918, XXIII, 173; 
191Q, XXIV, 405) confirm these results. Noguchi (Jour. Exper. Med., 1918, XXVIII, 
297) maintains that the optimum temperature is 37°C. but that maximum reactions may be 
reached at temperatures not lower than 23°C. Noguchi's results apply only to the test 
in which acetone-insoluble antigen is used, although the results of Smith and MacNeal 
show that fixation with this antigen is more specific by the "ice box method." See, also, 
Berghausen, Jour. A. M. A., 1919, LXXII, 996; Hitchcock, U. S. Naval Med. Bull., 1919, 
XIII, 740; Wile and Hasley, Jour. A. M. A., 1919, LXXII, 1526; Owen and Martin, Jour. 
Lab. and Clin. Med., 1920, V, 232. 



692 DIAGNOSTIC METHODS 

no hemolysis will occur in the tubes containing syphilitic serum. This lack of 
hemolysis, partial or complete, is, of course, a positive reaction. 

Citron^ advises the use of two tubes for serum and antigen, one tube con- 
taining 0.2 c.c. of serum and 0.2 c.c. of antigen as given above, while the other 
tube contains o.i c.c. of serum and o.i c.c. of antigen. In this way the results 
may be represented somewhat more quantitatively, as the degree of inhibition 
of hemolysis varies according to the amount of syphilitic antibody present. 
If this amount be large, complete inhibition occurs in the tube containing 0.1 
c.c. of serum and 0.1 c.c. of antigen as well as in tube No. i, the result being 
expressed as + + ++. If inhibition is incomplete in tube 2 but complete in 
tube I, the result is + ++. Both of these degrees are strongly positive 
reactions. If tube 2 is completely hemolyzed while tube i is completely 
inhibited, the result is + + . Incomplete inhibition in tube i and complete 
hemolysis in tube 2 is expressed as +. These two latter results are classed 
as weakly positive reactions. If tube i shows doubtful inhibition while 
tube 2 shows complete hemolysis, the result is ± . Complete hemolysis in 
both tubes is, of course, a negative result.^ 

Noguchi's Modification. 

This is not to be confused with Noguchi's test in which a human hemolytic 
system is used. The modification here suggested uses a different antigen and 
different quantities of the same reagents as employed by Wassermann. This 
is the method, followed in the writer's laboratory, when the sheep hemolytic 
system is used, a control being run with Sachs' antigen or a specific watery 
extract as antigen in cases showing affections of the central nervous system. 

1. Set up the double row of tubes as in the preceding test using 0.2 and 0.1 
c.c. of serum to be tested. 

2. To all tubes add 0.1 c.c. of an undiluted complement. 

3. To front row only, add 0.1 c.c. of acetone-insoluble antigen. 

Make up volume of each tube to 1.5 c.c. with physiologic salt solution. 
Mix contents thoroughly and incubate at 37°C. for one hour. 

4. Add to each tube i c.c. of antisheep amboceptor (two units). 

5. Add to each tube 0.5 c.c. of a 10 per cent, suspension of sheep corpuscles. 
The total volume is now 3 c.c. Mix contents thoroughly and incubate at 

37°C. for two hours. Allow to stand four to six hours at room temperature 
and read results. The interpretation is as given above. 

Drop Method. 

This is the method of Landsteiner, Miiller and Poetzl and is frequently 
employed. The writer can not convince himself that this method is an 
accurate one owing to the possible variations in size of the drops, which are 
delivered from a capillary pipet. 

^ Handbook of Kraus and Levaditi, 1908, II, 1105. 

2 Ivy (Jour. Am. Med. Assn., 191 2, LIX, 432) advocates the use of the Duboscq color- 
imeter as a means of estimating the exact degree of hemolysis, the control tube of each case 
being taken as the standard of 100 per cent. Ninety per cent, and over of hemolysis is 
regarded as negative. See Kaplan (New York Med. Jour., 1913, XCVII, 11 72; Ibid., 
1913, XCVIII, 157); and Kaplan and McClelland (Ibid., 1012) for the determination of 
amino nitrogen as a control. 



THE BLOOD 693 

1. Set up the double row of tubes as in the preceding tests and add to each 
10 drops of physiologic salt solution. 

2. Add I drop of serum to each of the two tubes used for the sera tested. 

3. To front row only, add 2 drops of antigen. 

4. To all tubes add i drop of complement. 

Mix thoroughly and incubate for one hour at 37°C. 

5. Add I drop (two units) of standardized antisheep amboceptor to each 
tube. 

6. Add I drop of a 50 per cent, suspension of sheep corpuscles to each tube. 
Incubate for two hours. Allow to stand at room temperature and read 

results. The method of interpretation is as above. ^ 

Method of Noguchi.^ 

It has been found that human serum contains a variable amount of natural 
antisheep amboceptor.^ This, of course, increases the effect of the immune 
antisheep amboceptor used in the tests previously described. One may, 
therefore, obtain a negative Wassermann test under these circumstances 
even though a large amount of complement is fixed. Noguchi has overcome 
this possible error by introduction of a human hemolytic system to replace 
the sheep hemolytic system of the original test. He employs, also, a smaller 
amount of reagents and uses either fresh or inactivated serum of the patient. 
If preferred, one may use larger quantities of patient's serum, than outlined 
by Noguchi; if so, the reagents are increased in the same proportion. This 
method of Noguchi is the one preferred by the writer and is the one followed 
as a routine in his laboratory, as it has been found somewhat more reliable 
than the original Wassermann test in obscure cases. ^ Sachs' antigen is used 
in every test. 

Collection of Serum. 

Noguchi obtains the patient's blood from a puncture on the ventral side of 
the last joint of the middle finger. The finger may be compressed by squeez- 
ing before the puncture and, afterward, massaged to press out the blood, 
this being collected in small tubes drawn out into capillary points. In 
the writer's laboratory the blood is obtained as given on page 686. 

Corpuscle Suspension. 

The suspension may be prepared with the blood of the patient or with that 
of the examiner. If with that of the former, it is especially important that 
every trace of serum be removed by careful washing. The standard amount 
of corpuscle suspension is i c.c. of a i per cent, or o.i c.c. of a 10 per cent, sus- 
pension for each tube. If the patient's blood is to be used, enough may be 
obtained for many tests as follows: Fill a graduated centrifuge tube to 9 c.c. 

1 See Mefford, Illinois Med. Jour., 1914, XXVI, 428.. 

2 Jour. Exper. Med., 1909, XI, 392; Serum Diagnosis of Syphilis, Phila., 1912. 

^ See Dexter and Cummer, Arch. Int. Med., 1912, IX, 605; also, Bailey, Ibid., 551. 

^ See Coca and L'Esperance (Arch. Int. Med., 1913, XI, 84) for a slight modification of 
theJNoguchi technic. See, also. Miller, Interstate Med. Jour., 1913, XX, 145; Thompson, 
Arch. Int. Med., 1913, XI, 512. See Cecil and Lamb (Arch. Int. Med., 1913, XI, 249) for 
the value of this test in testing the serum of cadavers. 



694 DIAGNOSTIC METHODS 

with 2 per cent, sodium citrate in physiological salt solution. Allow the 
blood to drop in until it fills the tube to 10 c.c. Mix and centrifuge. Pour 
off the supernatant fluid and fill up to 10 c.c. with fresh salt solution. Mix 
thoroughly and again centrifuge. This may be repeated two or three times, 
in order to insure removal of every trace of serum. The deposited corpuscles 
may now be suspended either in 100 c.c. of salt solution (making a i per cent, 
suspension) or in 10 c.c. (giving a 10 per cent, suspension). This procedure, 
while thoroughly reliable, is not followed by the writer. It is our custom to 
obtain the corpuscles from a puncture of the finger of the worker, allowing 
3 drops of blood to fall into 12 c.c. of salt solution in a centrifuge tube. This 
gives, approximately, a i per cent, suspension. The washing is done as 
above. This latter method enables us to obtain fresh suspensions whenever 
desired. 

Hemolytic Amboceptor. 

This is prepared, as previously outlined, by injecting a rabbit, intraperi- 
toneally, with increasing doses of washed human corpuscles, instead of with 
sheep corpuscles as in the Wassermann method. After the animal is immu- 
nized, the blood is collected and allowed to coagulate. The serum is then 
withdrawn and titrated as previously described. 

Amboceptor Paper. — This has been introduced by Noguchi to simplify the 
test and to enable those not in close touch with clinical laboratory facilities to 
perform the test. While this paper may be used, the writer advises those do- 
ing any number of these tests to place their reliance more in the liquid ambo- 
ceptor. The serum, obtained as above, is poured over sheets of filter-paper 
(Schleicher and Schiill's No. 597), cut into squares of 10 x 10 cm. Allow all 
the sheets to become evenly wet and absorb the excess with another sheet of 
paper. These are then dried at room temperature by placing each square 
separately upon a clean sheet of unbleached muslin. When dry, the sheets 
are cut into strips 5 mm. wide and are then standardized as follows: Take a 
series of tubes containing i c.c. of the i per cent, erythrocyte suspension and 
add to each tube 0.02 c.c. (as one unit) of complement. Now add the strips 
in increasing lengths, as i mm., 2 mm., 3 mm., etc., and incubate for two 
hours. The shortest strip causing complete hemolysis in this time represents 
one unit of amboceptor. These strips are then marked into sections of twice 
this length (two units) and cut off when the actual test is to be made. The 
papers should be kept dry and sealed. The papers on the market are quite 
variable and are not always to be rehed upon. As previously stated, these 
papers should be used only when the liquid amboceptor is not at hand. 

Complement. 

Guinea-pig serum is used as complement in this system as in the others. 
The dilution is 40 per cent, made by mixing i c.c. of complement with ij^^c.c. 
of physiologic salt solution. The method of obtaining it has been given above. 
Complement paper may be prepared but it is very unsatisfactory and should 
not be used if the liquid form can possibly be obtained. 



THE BLOOD 



695 



ORIGINAL WASSERMANN TEST 



Unknown serum . 
Unknown serum . 
Positive control . . 
Positive control. . 
Negative control. 
Negative control. 
Anticomplementary 

control. 
Hemolytic control. . 
Antigenic control. . . 



.5 D HI 

tii 

O hn 3 



C.C. 

0.2 
0.2 
0.2 
0.2 
0.2 

O. 2 



C.C, 

I 
I 
I 
I 
I 
I 
I 




«P^ 






1^ 

rt cfl 



s| 



Hemolysis 



Depends on sera. 
Complete. 
None, 
Complete, 
Complete, 
Complete, 
Complete, 

Complete. 
None. 



NOGUCHI'S MODIFICATION (SHEEP HEMOLYTIC SYSTEM) 




w 

C 

1 

3 


all 
S3l 


c 

1 

rt 

< 


Make up volume of each tube to 1.5 c.c. with salt 

solution. Mix contents and incubate at 

37° for one hour 


Ten per cent. 

suspension of 

sheep corpuscles 


Antisheep 
amboceptor. 
Two units 


"^1 

1! 

u ^ 

n 

i>o 

»-^ 

3 3 
^^ 


Hemolysis 


Unknown serum , , . 

Unknown serum 

Unknown serum 

Unknown serum . . . , 

Positive control 

Positive control 

Negative control. . . . 

Negative control 

Anticomplementary 

contro . 
Anticomplementary 

control. 
Hemolytic control. , 


c.c;-- 

0.2 
0.2 
0.1 

j 0.1 
0.1 

1 0.1 

1 0-2 
0.2 




C.C. 
O.I 
O.I 
O.I 
O.I 
O.I 
O.I 
O.I 
O.I 
O.I 

O.I 

O.I 


c.c, 

O.I 
O.I 
O.I 

■o.V 



0.2 

O.I 


c,c. 

0.5 
0.5 

o.S 

0.5 

0.5 

o.S 

0.5 . 

0.5 

0.5 

0.5 

o.S 


C.c. 
1 


Depends on sera. 

Complete, 
Depends on sera. 

Complete, 

None, 

Complete. 

Complete. 

Complete. 

Complete. 

Complete. 
Complete. 











NOGUCHI'S MODIFICATION (HUMAN HEMOLYTIC SYSTEM) 


; 


B 

3 

u 

B 
3 


-H" '0 

rt bfl"* 

all 
(3«| 


< 


rt"| 
(U jH 3 

I'rtS 

li 


a 


J 

So 

f 



+^ 
X 


p 


12 

II 

rot; 

m 

.S-2 

x< 


Hemolysis 


Unknown serum . , , , 
Unknown cerum .... 

Positive control 

Positive control 

Negative control. . . . 
Negative control, . . . 
Anticomplementary 

control. 
Hemolytic control. . . 


c.c, 
0.02 
0.02 
0.02 
0.02 
0.02 
0.02 


C.C. 
O.I 
O.I 
O.I 
O.I 
O.I 
O.I 
O.I 

0. 1 


C.C, 
O.I 

O.I 

O.I 



O.I 


C.C, 

I 


2 units 
2 units 
2 units 
2 units 
2 units 
2 units 
2 units 

2 units 


Depends on sera. 
Complete, 
None, 
Complete, 
Complete. 
Complete. 
Complete. 

Complete. 















696 DIAGNOSTIC METHODS 

Antigen. 

The antigen, used in this system, is prepared as previously described (p. 
685). The stock solution is a 3 per cent, methyl alcohol solution of the ace- 
tone-insoluble lipoids. From this a 0.3 per cent, emulsion is prepared for the 
test by mixing i c.c. of this stock solution with 9 c.c. of physiologic salt solu- 
tion. It seems advisable always to control this antigen by running a control 
test with a watery extract of specific organs, and of Sachs' antigen, especially 
if an affection of the central nervous system obtains. 

Technic. 

1. Arrange the double row of tubes with same controls as in the other 
tests. Place in all tubes i capillary drop (0.02 c.c.) of the fresh serum to be 
tested. If the serum has been inactivated use 4 drops (0.08 c.c). One may 
use 0.2 ex. of fresh (not inactivated) cerebrospinal fluid. 

2. Add to each tube o.i c.c. of 40 per cent, complement, or in a great 
emergency two units of complement paper. 

3. To the tubes in front row of each set, add o.i c.c. of antigen emulsion. 

4. To both tubes of each set, add i c.c. of i per cent, washed human 
corpuscles. 

Mix thoroughly and incubate for one hour at 37°C. 

5 . Add to each tube 2 units of antihuman amboceptor in liquid or paper form. 
Mix and incubate for two hours at 37°C. Allow the tubes to stand at 

room temperature for a few hours and read results. 

The results of this test are interpreted as in the preceding ones. This 
human system is more adjustable and reliable than is the sheep system. 
Noguchi has pointed out certain variable factors which may obtain and which 
may require a proper adjustment to insure proper results. These variables 
are, it is to be understood, greater in the sheep system, and are far less easily 
controlled. I quote directly from Noguchi. (i) One sometimes meets with 
instances in which hemolysis is complete within 10 to 20 minutes, and in 
which the positive control tubes with antigen undergo, sooner or later, gradual 
hemolysis. Such rapid progress of hemolysis at first mentioned is a sign of 
imperfect reaction. If the test is properly made, hemolysis proceeds gradu- 
ally, and is complete in the water-bath within half an hour or thereabout. 
The causes of this accelerated hemolytic process are either an abnormally 
weak resistance of the blood corpuscles, or an exceptionally high activity or 
insensitiveness to fixation of the complement employed; or it may be the re- 
sult of all these acting together. ^ It happens occasionally that the serum of 
certain guinea-pigs contains an abnormally active complement. In order to 
establish this point, and thus to remove this source of error, one has only to 
make the test with a quantity of complement which corresponds exactly to 
two complement units. (2) There are sometimes encountered instances in 
which hemolysis remains incomplete even in the control tubes in which there 
is no antigen. Here the causes of the imperfect reaction are found either in 
the weakness of the complement, or the amboceptor used, or both.^ Usually 

^ See Bailey, Jour. Exper. Med., 191 2, XV, 470. 

2 See Barratt, Jour. Path, and BacterioL, 191 2, XVI, 363. 



THE BLOOD 697 

the cause is the weakness of the complement, which, owing to its great lability, 
is likely to deteriorate. The activity of the amboceptor is far less subject to 
external influences which bring about its deterioration, and it is therefore ex- 
tremely rare to find that the imperfection in the reaction arises from this 
source. In testing several specimens of serum at one time it happens oc- 
casionally that some specimens are slower in completing the hemolytic re- 
action than others. The cause of this slowness is not present in the comple- 
ment or amboceptor, but in the specimens themselves. ^ In such cases the 
specimens are found to contain anticomplementary substances which react 
with and reduce the activity of the complement. To remove this source of 
error, it is necessary to heat the serum to 55°C. for 20 minutes and use four 
drops for the test. The difficulty may be obviated in some cases by collecting 
specimens of serum to be tested just before meal-time because the anticom- 
plementary substance is closely associated with the absorption of the chyle 
into the circulation soon after the meal. (3) The quality and quantity of the 
antigen can also be sources of error. If one uses poor antigen, either there 
will be no positive reaction at all, or weak positive reactions will be entirely 
overlooked. If, on the other hand, an excessive amount of unfractionated 
crude antigen is employed, certain nonspecific weak reactions may become 
manifest, or a false positive reaction even may be obtained, as the result of 
the action of anticomplementary substances sometimes contained in prepara- 
tions of the antigen. These sources of error can be entirely excluded by 
choosing an antigen that has been carefully prepared and standardized. ^ 

^ See Bronfenbrenner and Noguchi, Jour. Exper. Med., 191 2, XV, 598 and 625; Olson 
(Jour. Lab. and Clin. Med., 191 6, I, 704) calls attention to the importance of what he 
styles this "Delayed Wassermann Reaction." McConnell (Ibid., 1919, V, 43) studies 
this reaction by examining the tubes every 15 minutes after placing the tubes, to which the 
hemolytic system was added, in a water bath at 37°C. He interprets as a "delayed" 
reaction, one in which the tube containing cholesterinized antigen showed no hemolysis at 
the end of 30 minutes but had completely cleared up at the next period of examination. No 
such delay is observed in the tube containing plain alcoholic antigen. About i per cent, 
of all Wassermann tests show this delayed reaction. 

2 For discussions of many obscure points regarding the nature of the reagents and the 
technic of the test see Addis, Jour. Infect. Dis., 191 2, X, 200; Dick, Ibid., 1913, XII, in; 
Lippmann and Plesch, Ztschr. f. Immunitatsforsch., 1913, XVII, 548; Baumann, Illinois 
Med. Jour., 1913, XXIV, 117; Burdick, Colorado Med., 1913, X, 135; Kolmer and Cassel- 
man, Jour. Med. Research, 1913, XXVIII, 369; Surface and Routt, Ibid., 441; Cummins, 
Ibid., 1913, XXIX, 23; Weil, Biochem. Ztschr., 1913, XL VIII, 347; Stillians, Jour. Cutan. 
Dis., 1913, XXXI, 316; von Gierke, Deutsch. med. Wchnschr., 1913, XXXIX, 692; 
Rabinowitsch, Ibid., 121c; Kolmer and Williams, Jour. Infect. Dis., 1913, XIII, 96; Spat, 
Biochem. Ztschr., 1913, LVI, 21. Kapsenberg, Nederl. Tijdschr. v. Geneesk., 1913, II, 
1007; Wassermann and Lange, Kolle and Wassermann's Handb., 1913, VII, 951; Lesser, 
Mtinch. Med. Wchnschr., 1914, LXI, 7o;Langer, Deutsch. med. Wchnschr., 1914, XL, 274; 
Olmstead, Med. Record, 1914, LXXXV, 341; Thiele and Embleton, Lancet, 1914, I, 526; 
Mcintosh and Fildes, Ibid., 860; Matzenauer and Hesse, Wien, klin. Wchnschr., 1914, 
XXVII, 319; Sternberg, Ibid., 545; Klein and Frankel, Miinch. med. Wchnschr., 1914, 
LXI, 651; Wechselmann, Berl. klin. Wchnschr., 1914, LI, 304; Blumenthal, Ibid., 1316; 
Nathan, Ibid., 1934; Stone, New York Med. Jour., 1914, XCIX, 1242; Grulee, Am. Jour. 
Med. Sc, 1914, CXLVIII, 688; Lyon and Eiman, Ibid., 885; Stephens, Arch. Diag., 1915, 
VIII, 126; Schlesinger, Med. Record, 1915, LXXXVII 61; Bana, Brit. Med. Jour., 1915, 
I, 587; Stitt and Clark, U. S. Naval Med. Bull., 1915, VIII, 410; Clark, Ibid. 411; Busila, 
Presse Med., 1915, XXIII, 364; Heidingsfeld, N. Y. Med. Jour., 1916, CIII, 673 ; Bogomolets, 
Ruusk. Vrach, 1916, XV, 1180; Hirschfelder, Jour. A. M. A., i9i6,LXVI, 1386; Fildes and 
Mcintosh, Lancet, 1916, II, 751; Kafka and Haas, Med. Klin., 1916, XII, 1312; Wehrbein, 
Jour. Infect. Dis., 1916, XIX, 806; Ottenberg, Jour. Immunol., 1916, II, 39; Smith and 
MacNeal, Ibid., 75; Wade, Jour. Med. Res., 1916, XXXIV, 113; Sellards and Minot, Ibid., 
131; Bronfenbrenner, and Schlesinger, Am. Jour. Syph., 1917, 1, 406; Thompson, Ibid., 555; 



69S DIAGNOSTIC METHODS 

Diagnostic Value of the Wassennann Test. 

As stated above, the Wassermann reaction, or any of its modifications, is 
not a specific immunity reaction. While the antibody is, presumably, 
syphilitic in origin, the antigen is a physiologic lipoid and not a product of the 
treponema pallidum. In other words, the complement fixation test, as ap- 
plied to the diagnosis of syphilis, is not the result of an interaction between 
homologous (specific) antigen and antibody. Nevertheless, we find that the 
reaction does have a certain specificity from the diagnostic, if not from the 
biologic, standpoint. There is no question but that the technic of the 
Wassermann Test should be standardized, so as to permit of more certainty 
in comparison of results of different workers. This is especially to be 
desired for those laboratories which conduct examinations for physicians 
in various parts of the country. It is, today, almost impossible to arrive 
at conclusions, when reports from different laboratories are at variance, 
owing to the variability of the technic employed. Attempts have been 
made to standardize this technic and suggestions have been offered by 
several workers, but up to the present time no satisfactory solution of 
this important phase of the work has been attained.^ 

As a general rule it may be stated, at the outset, that a positive Wasser- 
mann test, all proper precautions and controls being made, indicates syphilis 
in practically all cases. It is true that certain nonspecific conditions, espe- 
cially leprosy and, occasionally, scarlet fever, give positive reactions, but the 
large majority of cases of other diseases, reported as giving positive results, 
are those in which syphilis has not been absolutely excluded. In such cases 
the syphilitic infection may be latent instead of the active cause of the trouble 
at the time of making the test, yet it must be reckoned with in deciding as 
P; to the value of this test from the diagnostic point of view.^ 

On the other hand, it is to be distinctly understood that a negative Wasser- 
mann test does not exclude syphilis, a fact that must be taken into considera- 
tion when the question of marriage is involved. Such cases must be judged 

Craig, Ibid., 802; Bartlett and O'Shansky, Ibid., 776; Jour. Lab. and Clin. Med., 1917, 

III, 118; Williamson, Ibid., 191 7, II, 202, 266 and 268; van Saun, Ibid., 191 7, III, 59; 

Huddleson, Jour. Immunol., 1917, II, 147; Sherwood, Jour. Infect. Dis., 1917, XX, 185; 

Callary, Jour. Lab. and Clin. Med., 1918, IV, 140; Reudiger, Jour. Infect. Dis., 1918, 
1^ XXIII, 533; Kolmer, Am. Jour. Syph., 1918, II, 739; Victors, Ibid., 758; Larkin, Cornwall, 

and Levy, Jour. Lab. and Clin. Med., 1919, IV, 571; Jeanselme and Bloch, Bull. Med. 
]■ . Paris, 1919, XXXIII, 533; Thomas and Simon, Canadian Med. Jour., 1919, IX, 1016. 

j- ^ See Stillians, Am. Jour. Syph., 191 7, I, 767; Ottenberg, Arch. Int. Med., 191 7, XIX, 

; 457; Emery, Lancet, 1918, II, 547; Kolmer, Am. Jour. Syph., 1919, III, i; Brown and Kol- 

j mer, Ibid., 8; Kolmer and Brown, Ibid., 170; Kolmer, Matsunami and Trist, Ibid., 407 

! and 513; Kolmer and Flick, Ibid., 1919, III, 541; Todd, Southern, Med. Jour., 1919, XII, 

r 667. 

i ^ See Kolmer, Jour. Exper. Med., 191 1, XIV, 235; Field (Jour. Am. Med. Assn., 191 2, 

LVIII, 1681) has called attention to the occurrence of positive Wassermann tests in cases of 
lead poisoning. Hilgermann, Deutsch. med. Wchnschr., 191 2, XXXVIII, 118; Gibson, 
Australasian Med. Gaz., 1913, XXXIII, 300. For other diseases showing a Wassermann 
see Jakobovics, Jahrb. f. Kinderhkde., 1914, XXIX, 215; Fletcher, Lancet, 1914,. I, 1677; 
Butler, U. S. Naval Med. Bull., 1915, IX, 51; Hesse, Wien. klin. Wchnschr., 1915, XXVIII, 

I 62; Sutherland and Mitra, Indian Jour. Med. Research, 1915, II, 984; Verdozzi and Urbani, 

! Policlinico, 1915, XXII, Med. Sec, 529; Prins, Nedul. Tijdschr. v. Geneesk., 1916, II, 1562; 

'• Sonntag, Deutsche Med. Wchnschr., 1916, XLII, 1577; Thomson and Mills, Lancet, 1919, 

CXCVI, 782. 



THE BLOOD 699 

largely by the clinical manifestations and history rather than upon the 
negative character of this test. Varying percentages of positive results in 
known syphilitic and parasyphilitic conditions have been reported by various 
workers. Some of the more enthusiastic ones claim that a negative Wasser- 
mann reaction means that the case is not syphilitic. This view, however, must 
be regarded as untenable in the face of the large numbers of negative results 
in the literature and in the daily experience of those working in this field. 
The following table, compiled from Boas^ and Noguchi^ shows the reported 
cases of syphilis with the percentages of positive results.^ 

Considerable discussion has arisen in the literature regarding the relative 
merits of the Wassermann and Noguchi systems. As a general rule it is 
found, by one running these two systems in parallel, that the positive results 

^ Die Wassermannsche Reaktion., Berlin, 1914. 

2 Serum Diagnosis of Syphilis, Philadelphia, 191 2. 

^ See Lucas, Am. Jour. Dis. Child., 1912, III, 259; Churchill, Ibid., 363; Kaplan, Med. 
Record, 1912, LXXXI, 1132; Bates, Arch. Int. Med., 1912, X, 470; De Buys, Am, Jour. 
Dis. Child., 1913, V, 65; Milne, Am. Jour. Med. Sc, 1913, CXLV, 197; Wolbarst, New York 
Med. Jour., 1913, XCVII, 378; Grindon, Davis, Greiner and Weiss, Interstate Med. Jour., 
1913, XX, 221; Sarateanu and Velican, Monatsschr. f. Geburtsh. u. Gynak., 1913, XXXVII, 
89; Balcarek, Med. Klin., 1913, IX, 1552; Eliasberg, Deutsch. Ztschr. f. Chir., 1913, 
CXXIV, 113; Blackfan, Nicholson and White, Am. Jour. Dis. Child., 1913, VI, 162; Holt, 
Ibid., 166; Richards, Jour. Am. Med. Assn., 1913, LX, 1139; Kaplan, New York Med. 
Jour., 1913, XCVIII, 308; Mcllroy, Watson and Mcllroy, Brit. Med. Jour., 1913, II, 1002; 
Heidingsfeld, Jour. Am. Med. Jour., 1913, LXI, 1598; Mensi, Gazz. d. osp., 1913, XXXIV, 
1032; Post, Boston, Med. and Surg. Jour., 1913, CLXIX, 777; Kaplan, Jour. Am. Med. 
Assn., 1913, LXI, 2214; Emery, Lancet, 1914, I, 223; Philippson, Policlinico, 1914, XXI, 
20; Russo, Riv. osped., 1914, IV, 173; Descomps, Grece, Med., 1914, XV, 37; Lesser and 
Klages, Deutsch. med. Wchnschr., 1914, XL, 1309; White, Therap. Gaz., 1914, XXXVIII, 
307; MacKinney, Ann. of Surg., 1914, LX, 309; Bahr, Illinois Med. Jour., 1914; XXV, 245; 
Corbus, Ibid., 1914, XXVI, 112; Craig, Jour. Am. Med. Assn., 1914, LXII, 1232; Thomas 
and Ivy, Am. Jour. Med. Sc, 1914, CXLVIII, 55; Southard, Boston Med. and Surg. 
Jour., 1914, CLXX, 947; Stone, Med. Record, 1914, LXXXVI, 545; Fordyce, New York 
Med. Jour., 1914, C, 597; Craig, Am. Jour. Med. Sc, 1915, CXLIX, 41; Krauss, Biochem. 
Ztschr., 1915, LXVIli, 48; Pontano, Policlinico, 1915, XXII, 41, 77 and 113; von Gonzen- 
bach, Cor.-Bl. f: Schweiz. Aerzte, 1915, XLV, 161 and 225; Wolbarst, Interstate Med. 
Jour., 1915, XXII, 109; Gradwohl, Southern Med, Jour., 1915, VIII, 477; Robertson and 
Klauder, Jour. Am. Med. Assn., 1915, LXIV, 199; Keyes, Ibid., 804; Vedder and Hough, 
Ibid., 972; Weisenburg, Ibid., 975; Haberman, Ibid., 1141; Heimann, Ibid., 1463; Wile and 
Stokes, Ibid., 1465; Glomset, Ibid., 1915, LXV, 682; Uhle and MacKinney, Ibid., 863; 
Moore, Ibid., 1980; Whitney, Ibid., 1986; Walker and Haller, Ibid., 1916, LXVI, 488; 
Haller, Ibid., 882; Kolmer, Ibid., 1435; Falls and Moore, Ibid., LXVII, 574; Gougerot, 
Paris Med., 1916, VI, 181; Lowrey, Am. Jour. Ins., 1916, LXXII, 601;, Buhman, 
Surg. Gyn. and Obs., 1916, XXIII, 284; McNeil, Southern Med. Jour., 1916, IX, 202; 
Courtney and Shropshire, Ibid., 205; Gordon, Arch. Pediat., 1916, XXXIII, 273; Uhle 
and Mackinney, Boston Med. and Surg. Jour., 1916, CLXXIV, 55; Med. Times, 1916, 
XLIV, 301; Simons, Jones and Goddard, Interstate Med. Jour., 1916, XXIII, 654; 
Graves, Jour. Immunol., 1916, II, 53; Moursund, Texas State Jour. Med., 1916, XII, 
435; Judd, Am. Jour. Med. Sc, 1916, CLI, 836; Warthin and Wilson, Ibid., CLII, 157. 
Snow and Cooper, Ibid., 185; Fordyce, Ibid., 469; DeBuys and Langford, Am. Jour. Dis. 
Child., 1916, XII, 387; Ladd, N. Y. Med. Jour., 1916, CIV, 952; Craig, Mil. Surg., 1916, 
XXXVIII, 286; Am. Jour. Syph., 1917, I, 192; Peterson, Ibid., 211; Knapp, Ibid., 772; 
Ball, Jour. A. M. A., 1917, LXVIII, 262; Slemons, Am. Jour. Med. Sc, 1917, CLIII, 212; 
Schmidt, Chicago Med. Rec, 191 7, XXXIX, 185; Kaplan, N. Y. Med. Jour., 191 7, CV, 
728; Gehrmann, Am. Jour. Pub. Health, 1917, VII, 964; Wassermann, Berl. klin. Wchn- 
schr., 1917, LIV, 105; Freudenberg, Ibid., 303; Heller, Ibid., 306; Scheel, Ugesk. f. Laeger, 
191 7, LXXIX, 43; Slack, Castleman and Bailey, Boston Med. and Surg. Jour., 191 7, 
CLXXVII, 180; Mayer, Berl. klin. Wchnschr., 1918, LV, 86; Freudenberg, Ibid., 620; 
Lambert, Olmstead, and Stuart, Proc N. Y. Path. Soc, 1918, XVIII, 61; Special Report 
No. 21, Medical Research Committee, National Health Insurance, London, 1918; Day and 
McNitt, Am. Jour. Syph., 1919, III, 595; Ravant, Presse Med., 1919, XXVII, 129. 



yoo 



DIAGNOSTIC METHODS 



Condition 



Number of cases 



Positive results 



Primary syphilis 

Secondary syphilis . . . 

Tertiary syphilis 

Early latent syphilis.. 
Late latent syphilis.. . 
Congenital syphilis.. . 
Cerebrospinal syphilis 
General paralysis. . . . 
Tabes dorsalis 



1, 060 

3,526 

1,212 

1,233 

1,520 

125 

64 

498 

360 



59.0 per 

90.0 per 

84.1 per 
51.0 per 
39.0 per 

5 per 

6 per 
I per 
o per 



94. 

47 
88, 
70. 



cent, 
cent, 
cent, 
cent, 
cent, 
cent, 
cent, 
cent, 
cent. 



are somewhat higher with the Noguchi system. The following table, taken 
from Noguchi, gives the figures for the cases reported, in which the two tests 
were made upon the same serum. 



Condition 



Number of 
cases 



Wassermann 
positive 



Noguchi 
positive 



Primary syphilis \ 208 j 88 per cent. 

Secondary syphihs j 669 1 92 per cent. 

Tertiary syphilis : 455 I 74 per cent. 

Latent syphilis 305 54 per cent. 

Congenital syphilis ! 79 | 98 per cent. 

Cerebrospinal syphilis 55 i 73 per cent. 



94 per cent. 
98 per cent. 
83 per cent. 
68 per cent. 
98 per cent. 
80 per cent. 



Effect of Treatment on the Reaction. 

Under the influence of a vigorous course of mercurials, a positive Wasser- 
mann test may be made to disappear. This must, however, not be inter- 
preted as meaning that the syphilitic infection has been cured. It is almost 
daily observed by those doing large numbers of these tests that a patient, 
having the test made while under active treatment with mercury, will fre- 
quently give a negative test; while the same patient may show a positive 
reaction if the mercury be withdrawn for two or three weeks prior to the test. 
On the other hand, we occasionally find cases in which the treatment has 
resulted in a positive Wassermann, prior tests having been negative. These 
facts must be borne in mind by those who may attempt to estabHsh a diagno- 
sis without full knowledge of the conditions under which the test is being 
made. Mercurial treatment may render the infection latent, so that a nega- 
tive reaction may obtain until later active manifestations appear and become 
associated with a positive test. This variability has been shown by Craig, ^ 
who reports 52 cases treated, with 32 positives and 20 negatives after treatment 
ranging from two weeks to twelve years. Patients should not have had any 
mercurial treatment for at least two to three weeks prior to the test if any 
reliance at all is to be placed upon it. Patients well treated give negative 
reactions, while those receiving inadequate treatment, no matter for how long 
a period, show positive results. The reaction is, unquestionably, affected by 
mercurial treatment, but some cases persist in giving a positive reaction in 

^ Jour. Exper. Med., 1910, XII, 726; Arch. Int. Med., 191 1, VIII, 395. See Nelson and 
Anderson, Jour. A. M. A., 1915, LXV, 1905. Craig and Nichols (Jour. Am. Med. Assn., 
191 1, LVII, 474) and Craig (Ibid., 1913, LX, 565) call attention to the influence of 
alcohol in converting a positive test into a negative one within 24 hours. 



THE BLOOD 70I 

spite of treatment. The reaction may return shortly after treatment, while 
it is not, by any means, settled that the disappearance of a reaction justifies 
one in stopping treatment. 

After treatment with ''606" (salvarsan, dioxy-dimino-arseno benzol), 
the results are variable. In promptly cured cases, a positive reaction may 
disappear within two weeks or it may require four to five weeks to obtain a 
negative result. A positive reaction may persist for considerable time and 
then disappear, reappearing again after an uncertain period. It has been 
definitely established that, if a patient be syphilitic, a negative Wassermann 
can be converted into a positive one by an injection of salvarsan, owing to the 
fact that the spirochete are killed and their toxic action is temporarily in- 
creased, the bodies concerned in the Wassermann test being thereby increased. 
This is the so-called ''Provocative Wassermann Reaction" and can be ob- 
tained in every stage of infection.^ The blood for the test should be taken 
before the injection as a control and then 24 and 48 hours after. Occasion- 
ally it is wise to take the blood one to two weeks after the injection also, 
in cases which are of doubtful nature. 

In deciding as to the proper criterion of a cure in any case a negative test 
following a positive one has little value. To establish a basis for the assertion 
of a cure, one should insist that the following points be met. One year with- 
out treatment, without any suspicious signs, with several negative Wasser- 
mann reactions and no positive ones, and with a negative provocative Wasser- 
mann reaction and luetin test at the end of the year.^ 

/3. Diseases Other Tharx Syphilis.^ 

The complement-fixation test is, theoretically, applicable to any in- 
fectious disease. Acute infections, however, do not show positive results 
until sufficient time has elapsed to permit of absorption of enough toxin to 
cause a systemic response to invasion, as shown by the presence of specific 
antibodies in the serum tested. Sub-acute and chronic cases offer the best 
chances for positive results. Practically, however, the tests have not been 
equally satisfactory in all types of infection or invariably reliable in the same 
t^'pe. 

The principle of the test is the same as described above no matter what the 
condition under investigation. The hemolytic system may be either sheep 
or human, the reagents used being as mentioned with the exception of the 
antigen employed. These antigens are polyvalent in character, being pre- 
pared from several different strains of the suspected organism. As different 
strains of the same organism often differ markedly one from another, the 
possibility exists of the serum of an infected patient fixing complement only 

^ See Stokes and O'Leary, Am. Jour. Syph., 191 7, I, 629. 

2 See Nichols, Jour. x^m. Med. i\ssn., 191 2, LVIII, 603; also, Craig and Nichols, Studies 
of Syphilis, Bull. 3, War Dept., Ofl&ce of Sur. Gen., Washington, 1913; Pusey, Am. Jour. 
Med. Sc, 1913, CXLVI, 497. Corbus (New York Med. Jour,, 1914, 0,472) reports a case of 
second infection, the Wassermann having been negative for one and one-half years. See 
King, Jour. A. M. A., 1916, LXVII, 1669; Trimble and Rothwell, Ibid., 1984, LeComte, 
Am. Jour. Syph., 1919, III, 106. 

^ See Ludke in Handbook of Kraus and Levaditi, Erganzungsband i, 1911, 518; Ztschr. 
f. klin. Med., 191 1, LXXII, 545. 



702 DIAGNOSTIC METHODS 

in the presence of closely allied strains. Hence, the more strains of the or- 
ganism in any given antigen the more certain will be the results of the test. 

The antigens are prepared as follows: Cultivate as many different strains 
as possible of the organism on appropriate media, such as agar slants or blood- 
agar slants, and wash the surface of each slant with 2 or 3 c.c. of a 0.9 per cent, 
solution of sodium chlorid, scraping the organisms from the media with a 
platinum loop. Pour off the suspension into a sterile tube and shake thor- 
oughly. • Kill the organisms by heating the tube in a water-bath at 6o°C. for 
one-half hour. Standardize the number of bacteria so that i c.c. of the sus- 
pension contains 1,000 million bacteria (see last chapter). The amount of 
antigen to be used in the tests must then be determined by titration just as 
described for the Wassermann test. 

Gonorrhea. 

In systemic gonorrheal infections -the complement-fixation test is be- 
coming of considerable importance. The technic is carried out as given above. 
The antigen should consist of at least twelve strains of gonococci. In the 
writer's laboratory the human hemolytic system is employed exclusively and 
the results have been constant and satisfactory. Others prefer the sheep 
system. The same precautions must be followed here as in the Wassermann 
test, every point in the technic being carefully controlled. 

A positive reaction indicates a focus of active gonorrheal infection in the 
system; while a negative result does not exclude this condition, owing to the 
variability in the antigens mentioned above. The influence of treatment 
upon this test is not at all clear, but it must be remembered that all cases 
treated with vaccines will show strong positive reactions. Just how long after 
such treatment is discontinued one may expect to obtain a positive result is 
still unsettled, so that the absolute determination of a cure must rest largely 
on the clinical findings. This is an important point, especially where marriage 
is contemplated, as the possibility of infecting another must be kept in mind. 
Non-infectivity cannot be predicated on a single negative fixation test.^ 

Attempts to use this method, as a diagnostic procedure, have been made 

in typhoid fever, tuberculosis,^ meningococcic infections, cholera, pertussis, 

^ See Schwartz and McNeil, Am. Jour. Med. Sc, 191 1, CXLI, 693; Swinburne, Arch. 
Diagnosis, 191 1, IV, 227; Schwartz, Am. Jour. Med. Sc, 191 2, CXLIV, 369; Schwartz and 
McNeil, Ibid., 815; Lenartowicz, Tygodnik lekarski, 191 2, VII, 368; Dermatol. Wchnschr., 
1912, LV, 1179; Lespinasse and Wolff, Ilhnois Med. Jour., 1913, XXIII, 26; Smith, Am. 
Jour. Dis. Child., 1913, V, 313; Finkelstein and Gerschun, Berl. klin. Wchnschr., 1913, L, 
1817; McNeill, Arch. Pediat., 1913, XXX, 657; McDonagh and Kline, Jour. Path, and 
Bacteriol., 1913, XVII, 559; Thomas and Ivy, Arch. Int. Med., 1914, XIII, 143; Smith, 
Am. Jour. Dis. Child., 1914, VII, 230; Williams, Interstate Med. Jour., 1914, XXI, 1198; 
Kolmer and Brown, Jour. Infect. Dis., 1914, XV, 6; Thomas, Ivy and Birdsall, Surg., 
Gyn. and Obs., 1914, XIX, 390; Arch. Int. Med., 1915, XV, 265; Irons and Nicoll, Jour. 
Infect. Dis., 1915, XVI, 303; Sormani, Nederl. Tijdschr. v. Geneesk., 1915, I, 1077; Uhle 
and MacKinney, New York Med. Jour., 1915, CII, 737; Irons and Nicoll, Trans. Chic. 
Path. Soc, 191 5, IX, 305; Asch and Adler, Miinch. Med. Wchnschr., 1916, LXIII, 73; 
Warden and Schmidt, Jour. Lab. and Clin. Med., 1916, I, 333. 

2 In tuberculosis the results of this test have been rather variable. Some reports are 
extremely enthusiastic, while others are of a doubtful nature. It would seem that the 
results with the antigen of Besredka (an autoclaved filtered culture of tubercle bacilli 
grown on a liquid medium composed of alkaline broth to which is added egg white and egg 
yolkj are the most reliable. However, even with this antigen, Bronfenbrenner reports as 
high as 20 per cent, of positive results with syphilitic cases, while Craig, on the other hand, 



THE BLOOD 703 

small-pox, infection with pyogenic cocci and other infections but the results 
have not been conclusive or reliable. Likewise it has been employed in the 
diagnosis of infection with various animal parasites, such as in echinococcus 
disease; helminthiasis in general and trichinosis. Here, also, the results are 
still too uncertain to warrant the routine use of this test.^ 

D. Abderhalden's Sero-diagnosis.^ 
I. Of Pregnancy. 

Abderhalden has recently introduced a biologic test for pregnancy, which, 
he assumes, depends upon the presence of certain ferments found in the serum 
of pregnant animals. This test appears to be fairly certain in its results and 
should prove invaluable to the gynecologist and obstetrician. 

It will be recalled that the injection of foreign protein into the system 
results in the production of certain well-established biologic properties in the 
serum of the animal so injected. Among these properties one finds the 
formation of precipitins, agglutinins, anaphylactogens, etc. Abderhalden has 
shown that the parenteral injection (subcutaneous, intraperitoneal or in- 
travenous) of foreign protein or carbohydrate brings about the appearance in 
the blood serum of proteolytic or amylolytic ferments, as an indication of the 
effort of the system to protect itself against the possible toxic effects of this 

shows that only one per cent, of syphilitics show positive resuKs in his hands, when this 
antigen is appHed. Of course, this may mean that many syphilitics are, also, infected with 
tuberculosis. Cook, in his work, calls attention to the fact that the bacterial antigens, 
as used in this test, are specific for the acid-fast group of bacterial infections but that they 
are not capable of sufficient differentiation for diagnostic purposes. He believes that the 
test is of some value in calling attention to unrecognized tuberculosis, but does not always 
indicate a clinically active process. At present, complement fixation in tuberculosis can 
not be placed on the same level as in syphilis, either from the diagnostic or prognostic point 
of view. See Besredka, Ztschr. f. Immunitatsforsch., 1914, XXI, 77; Bronfenbrenner, 
Ibid., 1914, XXIII, 2 and 221; Arch. Int. Med., 1914, XIV, 786; Stinson, Bull. loi, Hyg. 
Lab., U. S. P. H. S., 1915, Craig, Am. Jour. Med. Sc, 1915, CL, 781; Cooper, Jour. Infect. 
Dis., 1916, XIX, 313; Bronfenbrenner, Kahn, Rockman, and Kahn, Arch. Int. Med. 
1916, XVII, 492; Miller, Jour. Lab. and Clin. Med., 1916, I, 816; Miller, Jour. A. M. A., 

1916, LXVII, 1519; Craig, Ibid., i9i7,LXVIII, 773; Burns, Slack, Castleman, and Bailey, 
Ibid., 1386; McCaskey, Am. Jour. Med. Sc, 191 7, CLIV, 648; Woods, Bushnell, and 
Maddux, Jour. Immunol., 191 7, II, 301; Bronfenbrenner, Jour. Lab. and Clin. Med., 

191 7, III, 50; Blumberg, Ibid., 397; Brown and Petroff, Am. Rev. Tuberc, 1918, II, 525; 
Lange, Ibid., 541; Stivelman, Ibid., 546; Wilson, Jour. Immunol., 1918, III, 345; von 
Wedel, Ibid., 351; Small, Ibid., 413; Stoll and Neuman, Jour., A. M. x\., 1919, LXXII, 
1043; Lewis, Am. Rev. Tuberc, 1919, III, 129; Young and Givler, Ibid., 476; Cooke, Jour. 
Infect. Dis., 1919, XXV, 493; Walthard, Cor. Bl. f. Schwerz, Aerzte, 1919, XLIX, 1577; 
Barnes and Bernton, Boston Med. and Surg. Jour., 1919, CLXXX, 38; Pritchard and 
Roderick, Jour. A. M. A., 1919, LXXIII, 1879; Moursund, Jour. Infect. Dis., 1920, 
XXVI, 85. 

^ See Kolmer, Arch. Int. Med., 1912, IX, 220; Hastings, Jour. Am. Med. Assn., 1913-, 
LX, 1208; Besredka and Manoukhine, Ann. de I'Inst. Pasteur, 1914, XXVIII, 569; Compt. 
rend. Soc. de Biol., 1914, LXXVI, 197; Hastings, Jour. Infect. Dis., 1914, XX, 52 and 72; 
Garbat, Am. Jour. Med. Sc, 1914, CXLVIII, 84; Friedlander and Wagner, Am. Jour. Dis. 
Child., 1914, VIII, 134; Olmstead and Luttinger, Arch. Int. Med., 1915, XVI, 6t, Winholt, 
Jour. Infect. Dis., 1915, XVI, 389; Howell, Ibid., 456; Nakajo and Asakura, Ibid., 191 5, 
XVII. 388 and 400, Kolmer and Strickler, Jour. Am. Med. Assn., i9i5,LXIV, 800; Olmstead 
and Povitsky, Jour. Med. Res., 1915, XXXIII, 379; Hirschfelder, Jour. A. M. A., 1915, LXV, 
2073; Tribondeau and Fichet, Bull, de I'Acad. Med., 1916, LXXVI, 256; Kolmer and Trist, 
Jour. Infect. Dis., 1916, XVIII, 20 and 64; Kolmer, Trist and Heist, Ibid., 274 and 88; 
Kolmer and Pearce, Ibid., 32; Kolmer, Ibid., 46; Dick and Dick, Ibid., 1916, XIX, 175 and 
638; Kolmer, Jour. Immunol., 1916, I, 51 and 59. 

2 Abderhalden, Abwehrfermente des tierischen Organisms, Berlin, 1914. See, also, 
Vaughan, Jour. Am. Med. Assn.,1914, LXIII, 365; Taylor and Hulton, Jour. Biol. Chem,, 
1915, XXII, 59; Adami, Jour. Canadian Med. Assn., 1915, V, 569. Pregl and de Crinis 
(Fermentf., 191 7, II, 58) have devised a micro Abderhalden test. 



704 DIAGNOSTIC METHODS 

non-hydrolyzed material. Such protective ferments are, he believes, specific 
and are never found in the serum of normal (non-injected) animals. The 
blood will be seen, therefore, to have acquired definite digestive properties 
apart from that resident to a more or less extent in the leucocytes. A further 
step, taken by Abderhalden and his pupils, has been the demonstration that 
substances, which are native to the system but foreign to the blood, may arise 
from physiologic or pathologic changes within the system itself and, after 
absorption into the blood, produce quite as definite a response as if the ma- 
terial were introduced from without. 

Schmorl and Veit have shown that syncytial cells and portions of the 
chorionic villi may become detached from the placenta and enter the mater- 
nal circulation, while other placentral products (of an unknown nature) pre- 
sumably also are either absorbed or washed into the blood current. As these 
substances are, in part at least, protein in nature, we should have, if the theory 
holds, a production of protective proteolytic ferments in the maternal blood, 
which are capable of digesting placental protein and are more or less specific. 
If, therefore, the serum of a suspected animal be treated with placental pro- 
tein, hydrolytic cleavage must occur with the formation of products capable 
of more or less easy detection, provided the serum contains such ferments. 
These are the principles which form the basis of the test as originated by 
Abderhalden. 

Since the introduction of this test an enormous amount of work has ap- 
peared dealing with its various phases.' The earlier publications seemed to 
confirm the finding of Abderhalden that specific ferments were present in 
certain conditions, capable of digesting only so-called homologous protein. 
Many of the studies were adverse, the results being attributed by Abderhal- 
den to faulty technic. During the last year numerous researches have been 
made, which seem to strike a somewhat discordant note. At the present time 
the status of this question may be summed up as follows : In every fresh serum, 
male or female, normal or abnormal, ferments are present in the blood 
which, under certain conditions, are capable of causing hydrolytic cleavage of 
protein. Unquestionably, these proteolytic ferments are found in increased 
amounts in cases of pregnancy, carcinoma and other conditions to which the 
Abderhalden test has been applied. These ferments are, however, not to be 
regarded as specific, in the sense that they digest only homologous protein 
added as a substrat, as it is very questionable whether they act at all on the 
tissue used in the Abderhalden test. Owing to the presence in the blood of 
normal antiproteolytic substances, these normal (and hence not defensive 
ferments) ferments cannot exert their activity. If, however, this so-called 
"antitrypsin" be removed from the blood (i) by extraction with chloroform 
or ether (Delezenne and Pozerski), (2) by inactivation by addition of 0.2 per 
cent, acetic acid to the serum (Opie), or (3) by adsorption with an organic 
substrat or substances such as kaolin, starch or agar (Plant, Kjaergaard and 
others), changes occur in the degree of dispersion of the colloidal system and 
the normal proteolytic ferments become free to act. Sera treated in any of 
the above ways will undergo self-digestion and liberate dialyzable products 



THE BLOOD 705 

capable of detection by the Abderhalden or other methods. The substrat 
(placental or other tissue) acts exactly in this way. Further investigations 
have shown that this reaction is similar in many respects to that of combina- 
tion of antigen and antibody as previously discussed, thus bringing the Ab- 
derhalden reaction into the realm of other immunity tests. According to 
Stephan, Hauptmann and, more recently, to Bronfenbrenner, the Abderhal- 
den test is possible only when complement is present. If complement is miss- 
ing, as when the serum is heated, any fresh serum will activate the test. 
From the later studies of Bronfenbrenner it seems probable that the reaction 
occurs in two stages: (i) the substrat (antigen) combines with the antibody, 
thus becoming sensitized; (2) this sensitized tissue then adsorbs the anti- 
trypsin, leaving the normal proteolytic ferments of the blood free to digest 
the protein of the serum. In other words, the Abderhalden test does not 
depend upon the presence of specific ferments which digest only homolo- 
gous tissue, but upon the presence of specific (probably) antibodies, which 
unite with the substrat and, in so doing, adsorb antitryptic bodies and render 
the normal proteolytic ferments active. In this sense the Abderhalden test is 
to be regarded as specific, as it is certain that positive reactions in suspected 
cases are far more numerous, under the most carefully controlled conditions, 
than are the negative findings.^ However, the specifi(^ity is far removed from 
that claimed by Abderhalden. ^ 

^ In recent years there has arisen a decided tendency, based upon an accumulation of 
experimental evidence, to ascribe various phenomena of immunity to variations in the 
colloidal dispersion of the serum proteins and lipoids. Bronfenbrenner (Jour. Lab. and 
Clin. Med., 1916, I, 573) asserts in regard to the Abderhalden test that "the element of 
specificity lies, not in the ferment itself, but in the mechanism of its activation." The 
combination of specific serum with its corresponding antigen is followed by a radical change 
in the degree of dispersion of serum colloids. See, also, Jobling and Petersen, Jour. A. M. 
A., 1916, LXVI, 1753. 

^ For discussions of the various points raised in this newer investigation see Gammeltoft, 
Ugeskr. f. Laeger, 1913, LXXV, 1247; Heilner and Petri, Miinch. med. Wchnschr., 1Q13, 
LX, 1530; Oeller and Stephan, Ibid., i9i4,LXI, 12, 75 and 425; Plant, Ibid., 238; de Waele, 
Ibid., 364; Flatow, Ibid., 468, 608, 1168 and 1500; Stephan, Ibid., 801; Hauptmann, Ibid.^ 
1 167; von Domarus and Barsieck, Ibid., 1553; Freund and Brahm, Ibid., i664;Lindig, Ibid., 
1668; Beumer, Ibid., 1999; Kjaergaard, Ztschr. f. Immunitats f. Orig., 1914, XXII, 31; de 
Waele, Ibid., 170; Herzfeld, Biochem. Ztschr., 1914, LXIV, 103; Ibid., 1915, LXVIII, 402; 
Wilhelm and Szandicz, Ibid., 1914, LXV, 219; Benech, Compt. rend. Soc. de biol., 1914, 
LXXVI, 361; Bettencourt and Menezes, Ibid., 1914, LXXVII, 102; Michaelis and 
Lagermarck, Deutsch. med. Wchnschr., 1914, XL, 316; Frankel, Ibid., 589; Bisgaard and 
Korsbjerg, Ibid., 1367; Mosbacher and Port, Ibid., 1410; Peiper, Ibid., 1467; Oeller and 
Stephan, Ibid., 1557; Friedmann and Sch5nfeld, Berl. klin. Wchnschr., 1914, LI, 348; 
Lange, Ibid., 785; Csepai, Wien. klin. Wchnschr., 1914, XXVII, 804; Franz, Arch. f. Gynak., 
1914, CII, 579; Heinemann, Monatsschr. f. Geburtsh. u. Gynak., 1914, XXXIX, 768; 
Adachi, Ztschr. f. Geburtsh. u. Gynak., 1914, LXXVI, 516; Jobling and Petersen, Jour. 
Exper. Med., 1914, XIX, 480; Ibid., 1914, XX, 37; Bronfenbrenner, Ibid., 1915, XXI, 
221 and 480; Jobling, Eggstein and Petersen, Ibid., 239; Jobling, Petersen and Eggstein, 
Ibid., 1915, XXII, 129 and 141; Echols, Jour. Am. Med. Assn., 1914, LXIII, 370; Eggstein, 
Ibid., 735; Falls, Ibid., 1172; Ibid., 1915, LXIV, 1898; Ibid., 1915, LXV, 524; Bronfen- 
brenner, Schlesinger and Mitchell, Ibid., 1915, LXV, 1268; Bronfenbrenner, Jour. Lab. and 
Clin. Med., 1915, I, 79; Jobling and Petersen, Bull. Johns Hopk. Hosp., 1915, XXVI, 356; 
Jobling, Petersen and Eggstein, Jour. Exper. Med., 1915, XXII, 568, 590, 597 and 603; 
Kabanow, Fermentforsch., 1915, I, 206; Falls, Trans. Chic. Path. Soc, 1915, IX, 303; 
Herzfeld, Deutsche Med. Wchnschr., 1915, XLI, 1151; Van Slyke, Vinograd-Villchur, and 
Losee, Jour. Biol. Chem., 1915, XXIII, 377; Hulton, Ibid., 1916, XXV, 163 and 227; 
Lampert, Katz, King, Kline, Kulasavicz, Jeffries and Kutzenberger, 111. Med. Jour., 191 6, 
XXX, 22; Fujimoto, Jour. Immunol., 1918,111,51; Robertson and Hanson, Ibid., 131; 
Linossier, C. R. soc. biol. Paris, 1918, LXXXI, 422; Yamakawa, Jour. Exper. Med., 1918, 
XXVII, 689 and 711. 
45 



li 



706 DIAGNOSTIC METHODS 

Two distinct methods of detecting the presence of these antibodies have 
been devised. The first, the optic method, is capable of very wide application 
to the diagnosis of different conditions and should prove extremely useful in 
solving many problems of great clinical importance. It requires, however, 
considerable skill and technical ability as well as rather expensive apparatus. 
The second, the dialyzation method, is much simpler both in technic and in 
necessary equipment. Both of these methods require the most assiduous 
attention to the various details given, if any dependence is to be placed upon 
the results of the tests. 

I. The Optic Method. 

The basis of this test is as follows: A solution of placental peptone in 
physiologic salt solution has a definite power of rotating the plane of polarized 
light. Likewise, the serum, both suspected and normal, has a similar action. 
The degree of rotation, however, of either remains permanent for some time 
at 37°C. If a solution of peptone and normal serum be mixed and the degree 
of rotation of this mixture determined, no appreciable change will be observed 
between the initial and final polarimetric readings. If, however, a solution 
of placental peptone, whose polarizing action is known, be treated with a serum 
containing the specific ferments above mentioned, digestion of the peptone 
occurs with the formation of products showing rotatory powers sufficient to 
change the initial rotation of the mixture to quite an extent. These changes 
may be observed at different intervals and interpreted as described later. 
Preparation of Placental Peptone. 

The fresh placenta is made blood-free by cutting it into small pieces and 
placing these under running water for about i5minutes.^ Dry the pieces be- 
tween folds of filter paper and place them in about five times their weight of 70 
per cent, sulphuric acid. Allow the mixture to stand for three days at room 
temperature and shake the container frequently. At the end of this time, 
place the container in ice-water and dilute the contents with 10 volumes of 
distilled water, stirring constantly. Remove the sulphuric acid by adding 
approximately the calculated amount of finely powdered barium hydrate and 
complete the precipitation with a known solution of this salt, stirring the mix- 
ture constantly. When the reaction of the mixture becomes neutral to lit- 
mus paper, filter off the barium sulphate. If the filtrate be turbid, refiltra- 
tion is necessary until a perfectly clear filtrate is obtained. The separation of 
the barium sulphate is much facilitated by the use of the large centrifuge, 
if such be at hand. Wash the precipitate with a large amount of cold water 
and combine the filtrate and washings. Test the mixture for both barium 
and sulphuric acid. If either be present, it must be removed. Now evapo- 
rate this barium- and sulphuric acid-free solution to dryness on the water-bath, 
under reduced pressure at a temperature not exceeding 40 or 5o°C. It is 
wise to test the evaporated material at several intervals for the presence of 
either barium or sulphuric acid, as these sometimes appear on concentrating 
the mixture. It is important that these be removed, as their presence will 

' For discussions of the chemical composition of the placenta see Harada, Acta Scholas, 
Med. Univ. Kioto, 1916, I, 283 and 291; Fenger, Jour. Biol. Chem., 1917, XXIX, 19. 



THE BLOOD 707 

result in further hydrolysis of the peptone and, in consequence, will lessen the 
value of the final product. A thick yellow syrup or a foamy mass remains 
after this evaporation. The product may be used in this form, but it is pre- 
ferable to purify it, if reliable results are to be invariable. 

This yellowish residue is dissolved in methyl alcohol with the aid of heat 
and the hot solution is poured into absolute ethyl alcohol. The peptone is 
thrown down as a yellow powder, which is soluble in water to a clear yellowish 
solution of weakly acid or amphoteric reaction. This powder is not hygro- 
scopic. A further purification is still advisable. Dissolve the above yellow 
powder in water up to a 5 per cent, solution and add lo per cent, solution of 
phosphotungstic acid as long as a precipitate forms. Filter and wash several 
times with water. Rub up this precipitate in a mortar with some water and 
twice its weight of barium hydrate. Filter again and remove the excess of 
barium from the filtrate with sulphuric acid. Filter off the barium sulphate 
and evaporate the filtrate to dryness under reduced pressure at 40 to 50°, as 
outlined above. This product is snow white and is permanent. 

It is absolutely essential for the successful application of the optic test 
that the placental peptone be as pure as possible. The same product is not 
always obtained by the above method, as the hydrolysis may proceed further 
than the peptone stage. Such products are unsuitable for the test. It is wise, 
therefore, to work as quickly and as carefully as possible with a large amount 
of placental substance, so that one may obtain an appreciable am.ount of 
placental peptone. If the product be found serviceable, it may be kept for 
years. A further point to be considered in the use of a prepared peptone is 
that the solution of this placental product must give absolutely no turbidity • 
with the serum to be tested. Such a finding is not infrequent, owing to the 
probable presence of precipitins in the product. Such a peptone cannot 
be used. 

A further important property of the prepared peptone must be its power 
of rotating the plane of polarized light. The degree must not be too small or 
the product will prove of little value. It will be seen, therefore, that the 
preparation of a serviceable and proper placental peptone is a matter of 
considerable difficulty and is paramount to the successful performance of 
the test. 

One may preserve the peptone, prepared as above, either in the solid state 
or in the form of a solution. The advantage of a solution is that one has on 
hand a large amount of material, which will give good comparative results, 
as the solution is permanent. Abderhalden formerly used solutions of 0.5 to 
2.5 per cent, strength. He now advises a 10 per cent, solution of the placental 
peptone in physiologic salt solution. This solution must be absolutely clear 
and colorless. If not, filter through thick paper or a Berkefeld. Preserve 
this clear solution by overlaying its surface with toluol. When required for the 
test, the solution is withdrawn by apipet dipping below the toluol. If care be 
taken to keep a layer of toluol over the solution, the stock material will be 
permanent for a long period. Should this solution become turbid at any time, 
the material should be thrown away and a new stock solution prepared as 



7o8 DIAGNOSTIC METHODS 

above. The optical activity of this stock solution must be tested before each 
test.i 

Obtaining the Serum. 
The serum of the patient is obtained as described under the complement- 
fixation tests, withdrawing lo to 15 c.c. of blood. Place the blood, as drawn, 
directly into a sterilized centrifuge tube, so that all cellular elements may be 
completely separated. For a successful test the serum must show no sign 
of the presence of cells. A further precaution to be taken is that no sign of 
hemolysis must be present in the serum. For this reason, the cells should be 
separated rapidly. It is wise to make the test on the same day on which the 
blood is taken, although, if the precautions above mentioned are observed, a 
delay of 24 to 48 hours does not materially affect the activity of the serum. 

Technic. 

Having prepared the 10 per cent, solution of placental peptone and having 
proven that it answers all the requirements above mentioned, place i c.c. of 
this clear solution (withdrawn by a pipet) in a small, clean, sterile test-tube. 
Add 2 c*c. of the clear suspected serum and shake the tube several times. Ex- 
amine the mixture carefully for any turbidity or precipitation. If any be 
observed, the test cannot be carried out. Add sufficient physiologic salt 
solution to the mixture to fill the i decimeter polarimetric tube (see below). 
This mixture with salt solution is preferably made in this way, rather than to 
add the salt solution after the peptone solution and serum have been placed 
in the polarizing tube. Any turbidity may be much more easily detected. 
Pour the above mixture into the i decimeter tube, whose mantle has been 
filled with water at 37°C. 

Carefully determine the initial rotatory power of the mixture, checking 
the readings several times and controlling them by subsequent ones after 5 
or 10 minutes. No change should be observed in these readings. Place the 
tube and its contents in the incubator at 37°C. and repeat the readings every 
hour for a few periods and then continue every 6 or 8 hours. Do not extend 
the investigation over more than 48 hours. Record all readings and interpret 
them as given below. 

Control tubes must be arranged as follows: (i) the peptone solution alone; 
(2) the suspected serum alone; (3) peptone solution plus normal serum; (4) 
peptone solution plus known positive serum; (5) peptone solution plus inacti- 
vated (heated to 6o°C.) suspected serum. In all of these controls the same 
conditions must be maintained and the same length of polarizing tube must 
be used as in the test itself. If any turbidity occurs in any of the control 
mixtures or tubes, these must be disregarded in interpreting the test as turbid 
solutions give variable results with the polarimeter. 

It goes without saying that this test requires the very best equipment 
possible. The cheap polariscopes are absolutely useless as they are not deli- 
cate enough to detect the fine variations given. The three-shadow instru- 
ment of Landolt-Lippich, made by Schmidt and Haensch, is especially to be 

^ The placental p3ptone may now be obtained on the market from the Hochst-Farb- 
werke Co., New York. 



THE BLOOD 709 

recommended. The polarizing tubes used are, preferably, the i decimeter 
tubes which are furnished with a mantle which may be filled with water at 
any desired temperature. If different length tubes are used in any of the tests, 
a correction must be made in order that comparative figures may be obtained. 

In performing this test, even to a greater degree than when the instrument 
is used in other work, much depends upon the ability of the worker to detect 
slight variations in the degree of the rotatory powers of the mixtures under 
investigation. The method is easy to learn, but the special sensibility toward 
such changes cannot be taught. Abderhalden cautions any one, who shows 
a working error of as much as 0.04° in his observations, against attempting to 
interpret the test. 

In reporting the result of this test, Abderhalden employs the following 
method. 

Deviations within 0.04° Negative 

Deviations between 0.05 and 0.1° Positive (+) 

Deviations between o.ii and 0.2° Positive (++) 

Deviations over 0.2° Positive (+ ++) 

2. The Dialyzation Method. 

This method is much more simple than the optic method, both as regards 
technic and apparatus. It must not be thought, however, that any less care 
is necessary in carrying out the details of the test. In fact, erroneous results 
are, perhaps, more easily obtained by careless manipulation when this method 
is employed. 

The basis of this method is the conversion of the colloidal non-dialyzable 
placental protein into dialyzable products through the activity of the fer- 
ments above mentioned. These products are, then, detected by simple 
color reactions in the dialysate. 

Preparation of Placental Albumin. 

Although Abderhalden does not regard the proteolytic ferments of the 
serum in pregnancy as absolutely specific for a given species of animal, he, 
nevertheless, insists that the protein preparations used in either of his 
methods be prepared from the placenta of the same species as that of the 
animal whose serum is to be tested. 

As autolysis proceeds fairly rapidly in placental tissue, the albumin should 
be prepared from placentas which are as fresh as possible. Remove the ex- 
ternal portions of the placenta, such as the membranes, and wipe away as 
much blood as possible. Cut the material into small pieces and wash for a 
short time in running water. ^ While this is being done, boil about 2 liters of 
water to which are added 2 drops of glacial acetic acid. Throw the washed 
bits of placenta into this boiUng water and boil for 5 to 15 minutes. Pour 
the mixture upon a loose quick-acting filter and boil the pieces again with a 
second portion of acidulated water for 5 to 15 minutes. Pour off this water 
and test with the triketohydrinden hydrate reaction given below. If a 
positive reaction obtains, the placental tissue must be again boiled with 

^ It is essential that all visible blood be removed as its presence will introduce a large error. 



yio 



DIAGNOSTIC METHODS 



acidulated water until a negative reaction occurs. The essential points in 
this process are rapid and complete coagulation of the placental albumin and 
the removal of all soluble dialyzable material which may react with the 
reagent mentioned above. ^ 

As soon as a negative result is obtained with the extractive water, pour 
the mixture into a wide-mouth flask, add some chloroform, overlay the fluid 
with toluol and stopper the flask; or place the material in several smaller 
glass jars and overlay with toluol. This placental albumin keeps almost in- 
definitely and may be removed from the containers as desired. It should be 
tested, from time to time, to show that it contains, in itself, nothing which 
may react with the reagents used in the later test.^ 
Obtaining the Serum. 

The serum is obtained by venous puncture as previously described, the 
blood (about lo c.c.) being drawn directly into a sterilized centrifuge tube. 
The cellular elements are separated as quickly as possible and the serum 
drawn off into a clean sterile tube. It is of especial importance in this test 
that the serum show no sign of hemolysis. As it has been shown that amino- 
acids are present in the blood during digestion and may, therefore, give a posi- 
tive reaction with triketohydrinden hydrate, it is wise to take the blood in 
the morning before breakfast in all cases. If such be not done, a less amount 
of serum must be used in the later test to compensate for this possible error. 
Selecting the Dialyzing Tubes. 

It is evident that this part of the preparation for the test is of extreme im- 
portance. The dialyzing thimbles must be permeable for peptone but notfor 
albumin. Unless these conditions obtain, the test is valueless. Not all of 
the thimbles, purchasable upon the market, are by any means available. 
Abderhalden advises the use of the diffusion shells No. 579A. of Schleicher 
and Schiill. Not all of these will answer the purpose. 

It is necessary, therefore, that all the dialyzing thimbles used in the test 
should have been previously tested and known to answer the above require- 
ments. As the thimbles are usually dry and hard when obtained, soak 
them in cold water for a few hours, place them in boiling water for a few 
seconds and keep them in water covered with toluol. 

To test these thimbles for their permeability for albumin, proceed as 
follows. Remove the thimble from the water and place in it 5 c.c. of serum 
or of a solution of egg albumin. Add a few drops of toluol to prevent bacterial 
action. Place 20 c.c. of distilled water in the dialyzing vessel and overlay 
this with toluol. This dialyzing vessel should be quite narrow, the distance 
between the wall and the thimble (when in place) being about }/^ cm. These 
vessels are kept plugged with cotton and are sterilized before use. Now sus- 
pend the thimble with its albuminous contents in the dialyzing tube in such 
a way that the fluid outside is as high or, preferably, a little higher than that 
within the thimble. Plug the vessel with cotton to prevent contamination 

^ Care must be taken not to add an excess of acetic acid to the water as this may interfere 
with the ninhydrin test and thus give rise to an appreciable error. 

2 Abderhalden recommends that this placental albumin be tested with ninhydrin before 
being used in any test. This is a vital point. 



' 



THE BLOOD 711 

and put the apparatus in the incubator at 37°C. for 18 to 24 hours. At the 
end of this time, test the dialysate (outside fluid) for albumin by the biuret 
or triketohydrinden hydrate reactions given below. Those thimbles giving 
negative results are retained to be tested for their permeability to peptone. 
The shells permitting the passage of albumin cannot be used in the test. 

Select those thimbles showing impermeability to albumin and wash them 
thoroughly in water. Place in them 5 c.c. of a i to 1000 solution of Witte's 
or, preferably, peptone from silk (peptone La Roche) and add sufficient toluol 
to cover the solution. Dialyze as above against 20 c.c. of distilled water, 
placing the apparatus in the incubator for 18 hours at 37°C. Those thimbles 
which permit the passage of peptone, as shown by the triketohydrinden hy- 
drate test, are then kept for use in these tests and the non-permeable ones are 
laid aside. The properly tested and selected thimbles are then preserved in 
water overlaid with toluol. 
Technic. 

Remove a few pieces of the coagulated placental albumin from the con- 
tainer, wash in distilled water and dry between filter paper. ^ Break this up 
into very small bits or grind up in a mortar. Weigh out three portions of 3-^ 
gram each. Place 3^^' gram in each of three tested dialyzing thimbles in such a 
way that none of the material touches or remains upon the top or outside of 
the shells. Carefully wash off the outside of the thimble by means of a 
stream of distilled water or hold it under running water. This is done to re- 
move any possible adhering placental albumin, which would vitiate the test 
later made. Now add to tube No. i, i to 1.5 c.c. of clear hemoglobin-free 
serum to be tested. This serum is withdrawn from its container by means of 
a sterile graduated pipet. Overlay the surface of the mixture in the thimble 
with toluol. This thimble is then placed in a sterile dialyzing tube, as de- 
scribed above, containing 20 c.c. of distilled water, which should stand slightly 
higher than the fluid in the thimble. Overlay the external fluid with toluol 
and plug the dialyzing vessel with cotton to prevent contamination. Place 
the apparatus in the incubator at 37°C. for 18 hours and then test the dialysate 
for peptone as outlined below. 
Controls. 

Charge thimble 2 with J^ gram of placental albumin and i c.c. of serum 
of a known positive control. Overlay with toluol and arrange as above. 

Charge thimble 3 with }/'2 gram of placental albumin and i c.c. of a known 
negative serum or with the inactivated (heated to6o°C.) serum used in the test. 

A further control should be run, using i c.c. of the serum alone without 
the addition of albumin, to prove that it does not contain any dialyzable sub- 
stances which will give the later reactions. 

The tests with all of these controls are carried out exactly as the test it- 
self, every precaution being taken to prevent the introduction of errors. The 
tests for cleavage products of albumin are made with one of the following 
tests, the latter being in some respects preferable. 

1 Test the material before use with the triketohydrinden hydrate reaction. Absolutely 
no trace of a blue coloration should obtain. 



^ 712 DIAGNOSTIC METHODS 

The Biuret Test. 

This was the test formerly employed by Abderhalden and has some 
advantages in that it does not react with certain dialyzable products not infre- 
quently present in the serum of normal subjects. It requires considerable 
care and skill in manipulation as well as in interpretation. Doubtful results 
are very frequent unless every precaution be taken. 

Remove about 10 c.c. of the dialysate by means of a pipet dipping below 
the toluol. Place this in a test-tube and add 5 c.c. of a 33 per cent, sodium 
hydrate solution. Mix by careful shaking and add very carefully, drop by 
drop, from a buret a very dilute (0.25 per cent.) solution of copper sulphate in 
such a way that a distinct contact ring is formed. If peptone is present, a 
violet-red to a pure red contact ring will be observed, sharply differentiated 
from the lower colorless and upper blue solutions. This is a positive reaction. 
A negative result is shown by the appearance of a distinctly blue ring. It is 
not the simplest matter to distinguish between the various shadings which 
occur, so that one must not make his decision without having had some ex- 
perience in differentiating the colorations obtained with pure albumin and 
peptone solutions. 

The Triketohydrinden Hydrate Reaction. 

This reagent occurs in colorless crystals readily soluble in water. It 
may be obtained from the Hochst-Farbwerke (Lucius & Briining) Company 

/COx 
lyijll under the trade name of "ninhydrin." Its formula is C6H4\' yC(0H)2. 

It is of especial importance that every precaution be taken to prevent error 
when this reagent is used, as reactions may rise from the presence of substances 
which are not at all associated with the hydrolytic products of protein 
material. This reagent is not by any means specific, even for albumin, pep- 
tone or amino-acids, although it was formerly believed that it reacted only 
with substances containing an amino and a carboxyl group, the former es- 
pecially in the a position. It has been shown that there is a larger number 
of compounds which are not in a chemical sense combinations with amino 
and carboxyl groups and which, nevertheless, give very characteristic re- 
actions. Among these we find: amines; amino-aldehydes; urea derivatives; 
amino-sulphonic acids; ammonium derivatives of certain organic acids, di- 
carbonyl compounds and halogen-aldehydes; ammonium compounds of 
thiosulphuric, oxy-sulpharsenic and selenic acids; ammonium formate, am- 
monium thio-lactate, etc. Of special importance is the fact that a very 
small amount of basic products of putrefactive origin will give a decided 
reaction (hence the importance of using every means to prevent decomposi- 
tion of the tissue) . 

Certain further precautions are essential. If any of the original placental 
albumin be left on the outside of the dialyzing thimble, a very obvious error 
will arise. If the serum used contains amino-acids, the amount must be 
determined by depth of color with the ninhydrin. One must avoid the 
presence of acid or ammoniacal fumes in the laboratory. Strong alkalies 



I 



THE BLOOD 713 

cause, in themselves, a coloration with the reagent, while dilute alkalies may 
decolorize the solution. Acids prevent the appearance of the blue color 
and will destroy the color already formed, even in the presence of a large 
amount of reacting material. It must be insisted, therefore, that the fluid 
to be tested be absolutely neutral. Further, all vessels and pipets must be 
absolutely clean and the water used must be free from bacteria.^ 

Remove 10 c.c. of the dialysate by means of a pipet dipping below the 
toluol and place this in a large test-tube. Add 0.2 c.c. of a i per cent, aqueous 
solution of triketohydrinden hydrate. Heat rapidly to the boiling point and 
keep the mixture boiling for one minute. If the reaction be negative, the 
solution remains colorless or becomes, at most, light yellow. If the reaction 
be positive, a deep blue color will appear either immediately or on allowing 
the tube to stand for a short time. After use wash the thimbles thoroughly 
in running water and then place them in boiling water for not over 15 
seconds. 

The reaction is carried out in the same way with the control tubes. Tube 
2 should show a distinct positive reaction, while tube 3 should give a negative 
result. The tube with serum alone should show a negative reaction, but oc- 
casionally it is positive owing to the presence of a large amount of amino- 
acids in the serum as drawn. If the controls are all positive, the test is, 
of course, valueless, as some factor has been imperfectly controlled. 

Bronfenbrenner's Modification. 

Using the known property of antibodies of uniting with antigen at low 
temperatures, which prevent the activity of the complement and, also, ex- 
clude the activity of the proteolytic ferment, Bronfenbrenner adds to 3^ gram 
of placental tissue, contained in a glass tube, 1.5 c.c. of the suspected serum. 
Place this in the ice box over night. The next morning the serum is poured 
off, the tissue is placed in a centrifuge tube and washed, by centrifugation and 
decantation, until all traces of the serum are removed. The sensitized tissue 
is then placed in a dialyzing thimble and 1.5 c.c. of fresh male guinea-pig 
serum or normal human serum are added. Follow the usual technic of Ab- 
derhalden from this point. A positive ninhydrin reaction, under these 
conditions, is almost absolute evidence of pregnancy. 

If the suspected serum, which has been in contact with the placental tissue 
in the ice box, be placed in a dialyzing thimble and kept at 37°C. for 18 hours, 
the dialysate will show a positive reaction due to the removal of the antitryp- 
sin and the later self-digestion of the serum protein. The untreated serum, 
used as a control, should show no ninhydrin reaction. 

^ See Ruhemann, Jour. Chem. Soc, Trans., 1910, XCVII, 1438 and 2025; Ibid., 1911, 
XCIX, 792; Ibid., 191 2, CI, 780; Abderhalden and Schmidt, Ztschr. f. physiol. Chem., 
1911, LXII, 37; Ibid., 1913, LXXXV, 143; Pearce, Jour. Am. Med. Assn., i9i3,LXI, 1456; 
Warfield, Ibid., 1914, LXII, 436; Halle, Lowenstein, and Pribram, Biochem. Ztschr., 1913, 
LV, 357; Neuberg, Ibid., 1913, LVI, 500; Herzfeld, Ibid., 1914, LIX, 249; Neuberg, Ibid., 
1914, LXVII, 56; Deetjen and Frankel, Miinch. med. Wchnschr., 1914, LXI, 466; Howe 
Biochem. Bull., 1914, III, 269; Emerson and Chambers, Jour. Lab. and Clin. Med., 1916, 
I, 752; Harding and Warneford, Jour. Biol. Chem., 1916, XXV, 319; Harding and MacLean, 
Ibid., 337; Retinger, Jour. Am. Chem. Soc, 1917, XXXIX, 1059. 1 



714 



DIAGNOSTIC METHODS 



Value of the Test. 

This test, applied by either one of the above methods, is extremely valu- 
able. Abderhalden's results, which have been definitely confirmed (see bibli- 
ography at the end of this discussion) , show that in the majority of cases in 
which the reaction was positive the patient has been proven to be pregnant; 
while negative reactions have been given only in non-pregnant cases. In 
other words, a negative result is definite, a point of distinction from a negative 
result with the complement-fixation test; while a positive result is evidence of 
the presence of specific elements within the blood (pregnancy), either at the 
time of the test or at least within a period which does not exceed two weeks 
(Schwarz). In the absence of a true pregnancy, hydatidiform mole and other 
pathologic conditions of the chorionic villi may be accountable for a positive 
reaction. A negative test should be considered as eliminating pregnancy. 

As this reaction is evidence of pregnancy, in most cases giving positive 
results (an error of lo per cent, being probable) ,it is especially interesting to find 
that it is positive from the middle of the second month of pregnancy on and 
disappears within lo to 15 days after the termination of the pregnancy. It is, 
therefore, of great value in early diagnosis as well as in the diagnosis of a, second 
pregnancy following so closely upon a previous one that other signs are not pres- 
ent. Differential diagnosis should be greatly improved by the use of this test 
and fewer laparotomies performed under the mistaken diagnosis of tumor. 
A further value of the test is found in the fact that medico-legal cases may 
be established on a firm basis of definite knowledge of the conditions present. ^ 

^ As this test is of so much importance I give a fairly full bibhography of the subject for 
reference. Abderhalden, Freund and Pincussohn, Prak. Ergeb. d. Geburtsh. u. Gynak., 
1910, II, 367; Abderhalden, Handb. d. biochem. Arbeitsmeth., 1911, V, 575; Ibid., 1912, VI, 
223; Abderhalden and Kiutsi, Ztschr. f. physiol. Chem., 1912, LXXVII, 249; Abderhalden, 
Ibid., 1912, LXXXI, 90; Ibid., 1912, LXXXII, 109; Miinch. med. Wchnschr., 1912, LIX, 
1 190, 1305, 1939, and 2172; Deutsch. med. Wchnschr., 191 2, XXXVIII, 2160; Abderhalden 
and Weil, Berl. Tierarztl. Wchnschr., 191 2, XXVIII, 665; Frank and Heimann, Berl. khn. 
Wchnschr., 191 2, XLIX, 1706; Franz and Jarisch, Wien. klin. Wchnschr., 191 2, XXV, 1441; 
Veit, Ztschr. f. Geburtsh. u. Gynak., 1912, LXXII, 463; Petri, Zentralbl. f. Gynak., 1913, 
XXXVII, 235; Schwarz, Interstate Med. Jour., 1913, XX, 195; Henkel, Arch. f. Gynak., 
1913, XCIX, 56; Lindig, Miinch. med. Wchnschr., 1913, LX, 288; Abderhalden, Ibid., 
411 and 462; Jellinghaus andLosee, Bull. Lying-in Hosp., New York, 191 2, IX, 68; Gutman 
and Druskin, Med. Record, 1913, LXXXIV, 99; Engelhorn, Miinch. med. Wchnschr., 
1913, LX, 587; Schlimpert and Hendry, Ibid,, 681; Freund and Brahm, Ibid., 685; Freund, 
Ibid., 700 and 763; Abderhalden, Ibid., 701 and 763; Lindig, Ibid., 702; Heimann, Ibid., 
915; Stange, Ibid. 1084; Riibsamen, Ibid., 1139; Schiff, Ibid., 1197; King. Ibid., 1198; 
Maccabruni, Ibid., 1259; Ann. di ostet., MiJano, 1913, I, 486; Abderhalden, Miinch. med. 
Wchnschr., 1913, LX, 1386; Lampe and Papazolu, Ibid., 1423 and 1533; Frank and Rosen- 
thal, Ibid., i425;Lichtenstein, Ibid., 1427; Heilner and Petri, Ibid., 1530 and 1775; Steising, 
Ibid., 1535; Frank, Rosenthal and Biberstein, Ibid., 1594; Abderhalden and Weil, Ibid., 
1703; SchHmpert and Issel, Ibid., 1758; Bruck, Ibid., 1775; Goudsmit, Ibid., 1775; Abder- 
halden and Schiff, Ibid., 1923; Plotkin, Ibid., 1942; Lampe and Fuchs, Ibid., 2112; Tschud- 
nowsky, Ibid., 2282; Behne, Zentralbl. f. Gynak., 1913, XXXVII, 613; Parsamow, Ibid., 
934; Mayer, Ibid., 1181; Porchownick, Ibid., 1226; Schlimpert, Berl. klin. Wchnschr., 
1913, L 1136; Rosenthal, Ibid., 1149; Gottschalk, Ibid., 1151; Veit, Ibid., 1241; Aschner, 
Ibid., 1243; Schafer, Ibid., 1605; Evler, Ibid., 1606; Ebeler and Lohnberg, Ibid., 1898; 
Williams and Pearce, Surg., Gynec. and Obst., 1913, XVI, 411; McCord, Ibid., 418; 
Judd, Jour. Am. Med. Assn., 1913, LX, 1947; Am. Jour. Med. Sc, 1913, CXLVI, 391; 
Pari, Gazz. d. osp., 1913, XXXIV, 727; Ekler, Wien. klin. Wchnschr., 1913, XXVI, 696; 
Jaworski and Szymanowski, Ibid., 922; Daunay and Ecalle, Compt. Rendu Soc. de biol., 
1913, LXXIV, 1 190; Sunde, Norsk Mag. f. Laegevid., 1913, LXXIV, 1234; Schlimpert, 
Brit. Med. Jour., 1913, II, 1003; Deutsch. med. Wchnschr., 1913, XXXIX, 1225; Jonas, 
Ibid., 1099; Naumann, Ibid., 2086; Evler, Med. Klin., 1913, IX, 1042 and 1086; Bauer, 
Ibid., 1797; Abderhalden, Monatschr., f. Geburtsh. u. Gynak., 1913, XXXVIII, 24; WolflF, 



THE BLOOD 715 

2. Of Other Conditions. 

The principles upon whicti Abderhalden's sero-diagnosis is supposedly 
based have been considerably altered by the newer researches. However, 
the method has found, and will continue to find, wide application in the 
diagnosis of varied conditions.^ Although the basic idea of the presence of 

Ibid., 394; Schwarz, Interstate Med. Jour., 1913, XX, 393; Jour. Am. Med. Assn., 1913, 
LXI, 484; Abderhalden, Gynak. Rundschau, 1913, VII, 467; Decio, Ibid., 436; Ann. di 
ostet., 1913, I, 198; Abderhalden and Fodor, Ztschr. f. physiol. Chem., 1913, LXXXVII, 
220; Abderhalden and Schijff, Ibid., 225 and 231; Gabastou and Widakowich, Semana Med., 
1913, XX, 813; Scherer, Berl. klin. Wchnschr., 1913, L, 2183; Williamson, Jour. Obst. and 
Gynec. Brit. Emp., 1913, XXIV, 211; Werner and von Winiwarter, Wien. klin. Wchnschr., 

1913, XXVI, 1841; Labusquiere, Ann. de Gynec. et d'Obst., 1913, XL, 664; Fraenkel, Berl. 
klin. Wchnschr., 1913, L, 2287; Kabanoff, Med. Obozr., 1913, LXXX, 312; Sabin, Presse 
med., 1913, XXI, 1016; Williams and Ingraham, Colorado Med., 1913, X, 364; Abderhalden, 
Miinch. med. Wchnschr., i9i3,LX, 2774;Lampe, Ibid., 2831; Meyer, Ibid., 2906; Wegener, 
Ibid., 1914, LXI, 15; Bronstein, Ibid., 74; Oeller, Stephan and Mayer, Ibid., 12 and 75; 
Behne, Zentralbl. f. Gynak., 1914, XXXVIII, 74; Akimoto, Ibid., 81 ; Diner, New York Med. 
Jour., 1914, XCIX, 478; Holmes, Chicago Med. Recorder, 1914, XXXVI, 213, 475 and 593; 
Schwarz, Jour. Am. Med. Assn., 1914, LXIII, 371; Lindemann, Ztschr. f. d. ges. exper. Med., 

1914, IV, 177; Guggenheimer, Ztschr. f. Chemotherap., 1914, III (2TI) 210; McLester, 
Am. Jour. Med. Sc, 1914, CXLVIII, 75; Pearce and Williams, Jour. Infect. Dis., 1914, 
XIV, 351; Pregl, Fermentforsch., 19 14, I, i; de Crinis, Ibid., 13; Abderhalden, Ibid., 20; 
Hirsch, Ibid., 33; Strauss, Ibid., 55; Paquin, Ibid., 58; Ross and Singer, Arch. Int. 
Med., 1914, XIV, 552; Ibid., 1915, XV, 724; Lange, Biochem. Ztschr., 1914, LXI, 193; 
Thar and Kotschnieff, Ibid., 1914, LXIII, 483; Salus, Ibid., 1914, LXV, 381; Parsamow, 
Ibid., 1914, LXVI, 269; Neuberg, Ibid., 1914, LXVII, 56; Halsen, Ibid., 277; Frankel, 
Ibid., 298; Thar and Kotschniefif, Ibid., 1915, LXIX, 389; Abderhalden, Deutsch. med. 
Wchnschr., 1914, XL, 268 and 401; Hirsch, Ibid., 270; AUmann, Ibid., 271; Pincussohn, 
Ibid., 425; Abderhalden, Ibid., 428; Lampe and Stroomann, Ibid., 635; Lampe, Ibid., 
1213; Melikjanz, Ibid., 1369; Nieszytka, Ibid., 1519; Hirsch. Ibid., 1560; Otto and Blumen- 
thal. Ibid., 1836; Eder, Ibid., 1838; Abderhalden, Miinch, med. Wchnschr., 1914, LXI, 
233 and 401; Voelkel, Ibid., 349; Singer, Ibid., 350; Lampe, Ibid., 463; Kammerer, Clausz 
and Dieterich, Ibid., 469; Swart and Terwen, Ibid., 603; Abderhalden and Grigorescu, Ibid., 
767; Abderhalden and Fodor, Ibid., 765; Schiff, Ibid., 768; Abderhalden, Holle and Strauss, 
Ibid., 804; Abderhalden and Paquin, Ibid., 806; Deetjen and Frankel, Ibid., 826; Abder- 
halden and Wildermuth, Ibid., 862; Rosenthal and Biberstein, Ibid., 864; Abderhalden and 
Ewald, Ibid., 913; Lichtenstein and Hage, Ibid., 915; Freymuth, Ibid., 916; Griesbach, 
Ibid., 979; Abderhalden and Grigorescu, Ibid., 1209; Herzfeld, Ibid., 1503; Abderhalden, 
Ibid., 1879; Jaffe and Pribram, Ibid., 2125; Nieden, Ibid., 2200; JafEe and Pribram, Ibid., 

1915, LXIl, 614; Puppel, Monatss'chr. f. Geburtsh. u. Gynak., 1914, XXXIX, 764; Fetzer, 
Ibid., 1914, XL, 598; Petri, Ibid., XLI, 309 and 388; Baumann, Ibid., 1915, XLII, 199; 
Wallis, Jour. Obs. and Gynec. of Brit. Emp., 1914, XXV, 53; Paine, Boston Med. and 
Surg. Jour., 1914, CLXX, 303; Ecalle, Arch. mens. d'Obs. et de Gynec, 1914, III, 257; 
Partos and d'Ernst., Ibid., 333; Jellinghaus and Losee, Am. Jour. Obs. and Dis. Women and 
Child., 1914, LXIX, 593; Kolmer and Williams, Ibid., 1915; LXXI, 899; Ibid., 1915, 
LXXII, loi; Grey, Bull. Johns Hopkins Hosp., 1914, XXV, 117; Echols, Wis. Med. Jour., 

1914, XIII, i; Stoner and Skeel, Cleveland Med. Jour., 1914, XIII, 392; Bullock, Lancet, 
II, 225; Gavronsky, Ibid., 1915, I, 119; Leitch, Brit. Med. Jour., 1914, II, 161 and 330; 
Hinselmann, Zentralbl. f. Gynak., 1914, XXXVIII, 258; Kjaergaard, Ibid., 264; Schott- 
laender. Ibid., 425; Primsar, Ibid., 438; Hussy, Ibid., 897; Gentili, Ibid., 11 59; Saxl, Berl. 
khn. Wchnschr., 1914, LI, 824; Brieger and Schwalm, Ibid., 839; Ebeler and Lohnberg, 
Ibid., 1915, LII, 319; Abderhalden, Med. Klin., 1914, X, 665; Lampe and Paregger, Ibid., 
725; Abderhalden and Grigorescu, Ibid., 728; Forster, Ibid., 772; von Graff and Saxl, Ibid., 
1387; Lindstedt, Hygiea, 1915, LXXVII, 833; Goldstone, Med. Rec, i9i4,LXXXVI, 67; 
Keitler and Lindner, Wien. khn. Wchnschr., 1915, XXVIII, 549; Goodman, Ann. Surg., 

1915, LXI, 149; Kafka, Fermentforsch., 1915, I, 254; Ewald, Ibid., 315; Bunzel and 
Bloch, Miinch. Med. Wchnschr., 1916, LXIII, 6; Rivas and Buckley, Jour. Med. Res., 

1916, XXXIV, 297; Oppler, Biochem. Ztschr., 1916, LXXV, 211; Welker and Falls, Jour. 
Biol. Chem., 1917, XXXII, 509, 515, 519, and 521. 

^ See Wolff and Frank, Berl. klin. Wchnschr., 1914, LI, 875; Gumpertz, Beitr. z. Klin, 
der Tuberk., 1914, XXX, 201; Gwerder and Melikjanz, Miinch. med. Wchnschr., 1914, 
LXI, 980; Ammenhauser, Ibid., 2000; Schultz and Grote, Ibid., 2510; Baeslack, Jour. Am. 
Med. Assn., 1914, LXII, 1002; Ibid., 1914, LXIII, 559; Luxenburg, Med. Khn., 1914, X, 
1 104; Varney and Morse, Michigan State Med. Jour., 1914, XIII, 515; Hegner, Cor.-Bl. f. 
schweiz. Aerzte, 1914, XLIV, 1292; Vladesco and Popesco, Compt. rend. Soc. de. biol., 
1914, LXXVII, 586; Smith, Jour. Infect. Dis., 1915, XVI, 319; Falls, Ibid., 466; Voelkel, 



7i6 



DIAGNOSTIC METHODS 



\\ 



i 



If 
III 

ijllii 



Specific ferments in the blood in pathologic conditions must, probably, be 
changed, yet the test appears to be somewhat specific in the sense that 
certain antibodies are probably present in the serum, which antibodies so 
sensitize the substrat that the antiproteolytic substance of the blood is 
withdrawn, thus leaving the protein of the serum free to undergo self-diges- 
tion by the normal proteolytic ferments of the blood. The possibihty un- 
questionably exists that the use of a definite substrat (homologous in nature) 
may enable us to detect and differentiate pathologic processes much more 
sharply than has hitherto been the case. Although the specific nature of this 
test, as originally advocated by Abderhalden, has been attacked and shown 
to be dependent on other factors than those originally assumed, yet it is 
certain that there is much diagnostic value to the test, whatever may be its 
explanation. Considerable study must, however, be made before we are in 
a position properly to interpret our findings in all cases. 

(a) Cancer. 

In no other condition is a certain and constant diagnostic test so much 
to be desired as in cancer. Many methods have been advocated but 
Abderhalden's test is by far the most promising. 

The technic of the test is as described above. As the protein material 
to be sensitized, cancerous tissue is employed, the material being cut into 
small bits and prepared exactly as is placental tissue. It would seem neces- 
sary to use homologous tissue in this work, that is carcinomatous or sarcoma- 
tous as the case may be. This point has not been definitely established but 
the results in any given case would, probably, be more specific. The dialyza- 
tion fluid is distilled water. Erpicum advises the use of a 2 per cent, solution 
of sodium fluorid as an antiseptic, but this is unnecessary if toluol is used 
freely as should be done. Sterility of all material and glass-ware is essential 
in this test. 

The results reported have been more than encouraging in that benign 
growths seem to give, almost constantly, negative results while the malignant 
forms show an extremely large percentage of positive findings. The writer 
has used this test frequently and has corroborated his findings by pathological 
examination of the tissues removed at operation in many of the cases. In 
some of the cases the results have been far from satisfactory.^ More detailed 
study is necessary before the value of this test in cancer is determined. 

Miinch. med. Wchnschr., 1914, LXI, 349; Wohl, Am. Jour. Med., Sc, 1915, CXLIX, 427; 
Oeri, Beitr. zur klin. der Tuberk., 1915, XXXIII, 211; Hippel, Fermentforsch., 1915, I, 
233; Lampe and Cnopf. Ibid., 269; Abderhalden, Ibid., 351; Smith and Cook, Jour. Infect. 
Dis., 1916, XVIII, 14; Elsesser, Ibid., XIX, 655; Hiissy and Herzog, Arch. f. Gynak., 
1916, CV, 142; Datta, Rif. Med., 1916, XXXIII, 204; Izabolinsky, Russk. Vrach, 1916, 
XV, 808. 

1 See Abderhalden, Miinch. med, Wchnschr., 1913, LX, 2385; Labbe, Gaz. med. de 
Nantes, 1913, 2 me. S., XXXI, 461; Frank and Heimann, Berl, klin. Wchnschr., 1913, L, 
631; Markus, Ibid., 776; Munzer, Ibid., 777; Epstein, Wien. klin. Wchnschr., 1913, XXVI, 
649; Deutsch and Kohler, Ibid., 1361; Erpicum, Bull, de I'Acad. roy. de med. de Belg., 
1913, XXVII, 624; Ludke, Gaz. d. hop., Paris, 1913, LXXXVI, 1064; von Gambaroflf, 
Miinch. med. Wchnschr., i9i3,LX, 1644; Brockman, Lancet, 1913, II, 1385; Abderhalden, 
Deutsch. med. Wchnschr., 1913, XXXIX, 2391; Hara, Ibid., ^559; Fried, Miinch. med. 
Wchnschr., 1913, LX, 2782; Schwarz, Am. Jour. Obst, 1914, LXIX, 54; Ball, New York 
Med Jour., 1913, XCVIII, 1249; Jour. Am. Med. Assn., 1914, LXII, 599; Halpern, 



THE BLOOD 717 

{b) Dementia Praecox. 

In the realm of nervous disorders differential diagnosis is not always as 
exact as could be desired. Abderhalden's test has been applied by many 
in such cases with excellent results. In this work the selection of the proper 
material for the substrat seems to be the matter of greatest importance. It 
is probable that testicular tissue should be used for male patients and ovarian 
tissue for female sufferers, although cortical tissue and thyroid have been 
used by some but positive results with the latter are few. The adoption of 
cortical tissue is highly improbable as differential diagnosis is not subserved 
by its use. General paralysis, epilepsy with dementia, and manic-depressive 
insanity all respond promptly to brain tissue but very rarely to testicular or 
ovarian material. A further point is that all syphilitic and parasyphilitic 
disorders seem to cause hydrolysis of cortical brain tissue but not of other 
organs. The antibodies found in the serum of these patients are, therefore, 
more or less specific, as a reaction with testicular or ovarian tissue is almost 
positive evidence of dementia praecox.^ 
E. Herman-Perutz Reaction. 

As the Wassermann test for syphilis is so complex and requires so much 
experience for its proper performance and interpretation, attempts have been 
made to find a serum reaction for syphilis which could be used by the general 
worker and which, at the same time, would give results comparable to those of 
the Wassermann test. Such a test is that of Herman and Perutz,^ which is a 

Mitt a. d. Grenzgeb. d. Med. u. Chir., 1914, XXVII, 340; Piorkowski, Berl. klin. Wchnschr., 
1914, LI 254; Frankel, Ibid., 356; Fasiani, Wien. klin. Wchnschr., 1914, XXVII, 267; 
Manoiloff, Ibid., 269; Oeller and Stephan, Miinch. med. Wchnschr., 19 14, LXI, 579 and 
583; Schawlow, Ibid., 1386; Weinberg, Ibid., 1617, 1685 and 1732; Frankel, Deutsch. med. 
Wchnschr., 1914, XL, 589; Hara, Ibid., 1258; Cytronberg, Mitt. a. d. Grenzgeb. der Med. 
u. Chir., 1914, XXVIII, 243; Heimann and Fritsch, Arch. f. Chir., 1914, CIII, 659; Lan- 
zarini, Gazz. d. osp., 1914, XXXV, 1057; Trubina, Deutsch. Zeitsch. f. Chir,, 1914, CXXXI, 
520; Fulchiero, Rif. med., 1914, XXX, 1261; Van Slyke and Vinograd, Proc. Soc. Exper. 
Biol, and Med., 1914, XI, 154; Dick, Jour. Infect. Dis., 1914, XIV, 242; Kahn, Arch. Diag., 
1914, VII, 356; Biehn, 111. Med. Jour., 1915, XXVII, 206; Goodman, Surg., Gyn. and Obs., 
1914, XIX, 797; Levin, New York Med. Jour., 1914, C, 621; Bullock, Lancet, 1915, 1, 223; 
Goodman and Berkowitz, Surg., Gyn. and Obs., 1915, XXI, 463; Ball, Jour. Am. Med. 
Assn., 1914, LXII, 599; Ibid., i9i4,LXIII, 1169; Lowy, Ibid., i9i5,LXIV, 1559; Levin and 
Van Slyke, Ibid., 1915; LXV, 945; Berghausen, Interstate Med. Jour., 1915, XXII, 228; 
Drummond, Biochem. Jour., 1916, X, 473; de Crinis and Mahnert, Fermentf., 1918, II, 103. 

1 See Fauser, Deutsch. med. Wchnschr., 1913, XXXIX, 304; Wegener, Miinch. med. 
Wchnschr., i9i3,LX, 1197; Schulz, Deutsch. med. Wchnschr., 1913, 39, i399;Lampe, Ibid., 
1774; Bundschuh and Roemer, Ibid., 2029; Fischer, Ibid., 2138; Fuchs, Miinch. med. 
Wchnschr., i9i3,LX, 2230; Binswanger, Ibid., 2321; Theobald, Med. Klin., 1913; IX, 1850; 
Berl. khn. Wchnschr., 1913, L, 2180; Beyer, Miinch. med. Wchnschr., 1913, LX, 2450; 
Kafka, Med. Klin., 1914, X, 153; Fauser, Miinch. med. Wchnschr., 1914, LXI, 126; 
Kastan, Deutsch. med. Wchnschr., 1914, XL, 319; Arch. f. Psychiat., 1914, LIV, 928; 
Grigorescu, Med. Klin., 1914, X, 418; Neue, Ibid., 1217 and 1259; Simon, Jour. Am. Med. 
Agsn., 1914, LXII, 1701; Sterne, 111. Med. Jour., 1914, XXVI, 327; Gehrmann, Ibid., 335; 
Holmes, Ibid., 332; New York Med. Jour., 1914, XCIX, 567; Chicago Med. Recorder, 
1914, XXXVI, 644; Ibid., 1915, XXXVII, 88; Bouman and Hasselt, Nederl. Tijdschr. v. 
Geneesk., 1915, I, 423; Mayer, Miinch. med. Wchnschr., 1915, LXII, 580; Sioli, Arch. f. 
Psychiat. u. Nervenkr., 1914, LV, 241; Obregia and Pitulesco, C. R. soc. bioL, Paris., 
1914, LXXVI, 47; Kafka, Miinch. Med. Wchnschr., i9i5,LXII, I3i6and 1355; Wegener, 
Fermentf orsch., 1915, I, 210; Parhon and Parhon, Ibid., 311; Colton, White and 
Stevenson, Jour. Nerv. and Ment. Dis., 1915, XIII, 259; Jour. Nerv. and Ment. Dis., 1916, 
XLV, 144; Singer and Quantz,- Arch. Int. Med., 1916, XVIII, 529; Falls, Jour. A. M. A., 
1916, LXVI, 22; Retinger, Arch. Int. Med., 1918, XXII, 234. 

2 Med. Khn., 1911, VII, 60. Landau (Wien klin. Wchnschr., 1913, XXVI, 1702; see, 
also, Presse med., 1914, XXII, 335, and Riv. osped., 1914, IV, 641) has introduced a test for 



7i8 



DIAGNOSTIC METHODS 



?!f 




IHII 



modification of an older test of Porges. Its simplicity should recommend 
it, especially as its results are usually definite and easily interpreted. 
For this test two solutions are used. 



Solution A. 



Sodium glycocholate, 

Cholesterin, 

95 per cent, alcohol, 



2.00 grams 
0.40 gram 
100.00 c.c. 



At the time of making the test, this solution is diluted with distilled water 
in proportion of i to 20. 

Solution B. 

A 2 per cent, aqueous solution of sodium glycocholate. 

This solution must be made up fresh at the time of making the test, as 
it is not permanent. Further, the solution is not a perfect one so that the 
bottle should be shaken before using. 

Technic. 
Withdraw the blood as for the Wassermann test. As only a small 
amount is needed, puncture of the ear or finger is usually sufficient. Col- 
lect the blood in a sterile tube, allow it to clot and centrifuge. The serum 
must show no hemolysis. Pipet off the clear serum. Although Herman and 
Perutz believed the test was better if the serum was inactivated by heating 
to 56°C. for one-half hour, later work has shown that this is not essential. 

syphilis, using as his reagent a i per cent, solution of iodin in carbon tetrachlorid. To 0.2 
c.c. of fresh serum he adds o.i c.c. of the above reagent, shakes the tube and allows the 
mixture to stand for four hours. Positive results are shown by a clear yellow serum above 
the decolorized reagent, indicating the binding of the iodin probably by the lipoids. If the 
serum is an opaque grayish white, the reaction is negative. While this test gives results in 
many cases which parallel those of the Wassermann test, yet it shows a large percentage of 
positive results in non-syphilitic cases. Further, a negative Landau test is of little value as 
many known S3T)hilitics give negative tests. This test has very little diagnostic value. See 
Golay, Rev. med. de la Suisse roman., 1914, XXXIV, 571; Villaret and Pierret, Presse 
med., 1914, XXII, 582; Correa, Brazil-Medico, 1914, XXVIII, 395; Kolmer, Jour. Am. 
Med. Assn., i9i5,LXIV, 1461 and 1966; Stillians, Ibid., 1964; Capello, Gazz. d. osp., 1915, 
XXXVI, 423; Recupero, Ibid., 854; Chiaravalloti, Pediatria, 1915, XXIV, 129. Bruck 
(Miinch. med. Wchnschr., 191 7, LXIV, 25) has introduced a test, which depends upon the 
difference in solubility in distilled water of a precipitate formed by dilute nitric acid in the case 
of syphilitic sera, as compared with normal sera. In this test 0.5 c.c. of clear corpuscle-free 
serum, which has not been heated, is placed in a large test-tube and 2 c.c. of distilled water 
added and the mixture gently mixed by shaking. The exact time being noted with a watch, 
0.3 c.c. of 25 per cent, nitric acid (sq. g. 1149, made by diluting 100 c.c. of C. P. Nitric acid 
with 225 c.c. of distilled water) is added and the mixture gently shaken. This is allowed 
to stand at room temperature for exactly 10 minutes. To the material in the tube, which 
should now contain a white precipitate, is added exactly 16 c.c. of distilled water The 
tube is closed with the finger and the contents mixed by inverting the tube three times, 
carefully so as not to cause foam. Allow this to stand 10 minutes and mix as before, 
after which the tube is set aside for ]/^ hour, when the result of the test is read. With 
normal serum, the precipitate is supposed to dissolve completely, the water being clear or 
slightly opalescent; with syphilitic sera, small flakes are formed, which show little tendency 
to dissolve and which settle out on allowing to stand. A well defined sediment is marked 
-f-; a slight one, ±; none, — . While this reaction parallels the Wassermann reaction in 
about 75 per cent, of cases of known syphilis, j^et the results are not sufficiently specific 
to warrant more than a confirmatory evidence. See Smith and Solomon, Boston Med., 
and Surg. Jour., 1917, CLXXVII, 321; Stillians, Jour. A. M. A., 1917, LXIX, 2014; Toy- 
ama and Kolmer, Jour. Cutan. Dis., 1918, XXXVI, 429; Boruttau, Ztschr. f. augew. Chem., 
1918, XXXI, 65; Terada,Kitasato Arch. f. Exper. Med., Tokyo, 1919, III, 123. 



THE BLOOD 719 

To 0.4 c.c. of the clear serum add 0.2 c.c. of the i to 20 dilution of solution 
A and 0.2 c.c. of solution B. Shake thoroughly, plug the tubes with cotton 
and allow them to stand not longer than 18 to 24 hours at room temperature. 
On examining the tubes, a positive result is indicated by a flocculent precipi- 
tate, while a negative result is shown by no precipitation. The results may 
be expressed as -f , + + , + + + depending on the degree of precipitation. 
This gives the results so that comparison with the Wassermann may be 
made easily. 

This test practically parallels the Wassermann test, except that in 
secondary syphilis the results are not quite so uniform with the former. The 
percentage of variation is greater than with the Wassermann test, so that our 
interpretation of the result would be guarded. In other words, a positive 
result indicates syphilis, while a negative result does not exclude syphilis. 
This reaction should find a place in the laboratory of every general worker 
who does not have facilities for performing the Wassermann test, but should 
not be relied upon to the exclusion of the Wassermann test.^ 

F. Coagvilo Reaction. 

This reaction, originated by Hirschfeld and Klinger,*^ is based on the 
observation that syphilitic serum delays or even prevents the coagulation of 
recalcified oxalate plasma by inhibiting the production of thrombin through 
interference with the activity of thrombokinase. It is to be recalled that 
coagulation of the blood is due to the formation of fibrin from fibrinogen 
through the influence of a ferment, thrombase (thrombin), which is present in 
the leucocytes and blood plates (and may be obtained in vitro by extraction 
of almost all tissues with alcohol) in the form of prothrombase. This 
latter zymogen is changed, through the influence of ionized calcium com- 
pounds, into thrombin. It has, further, been shown that thrombin consists 
of two substances: namely, (i) the serozyme or thrombogen, a protein con- 
stituent of the plasma and (2) the cytozyme or thrombokinase, which belongs 
to the group of lipoids (largely lecithins). This cytozyme, therefore, it will 
be seen, is essential to the formation of a coagulum. A simple procedure 
would, of course, be to test a fluid by the addition of fibrinogen solutions. 
However, such solutions are unstable, so that, as Bordet and Delange^ have 
shown, we may employ oxalate plasma instead. This latter has the advan- 
tage that it will keep for some time and that its addition prevents any further 
formation of thrombin, as the calcium of the serum is precipitated by the 
oxalic acid radical in the form of calcium oxalate. 

- ^ See Gammeltoft, Hospitals tid., 191 2, V, 471; Deutsch. med. Wchnschr., 191 2, XXX- 
VIII, 1934; Jensen and Feilberg, Hospitalstid., 1912, V, 493; Feilberg, Ibid., 973; Ipsen 
and Helweg, Ibid., 1341; Thomsen and Boas, Ibid., 1152; Jensen, Ibid., 1516; Pontoppidan, 
Ugesk. f, Laeger, 191 2, LXXIV, 1377; Jensen and Feilberg, Berl. klin. Wchnschr., 1912, 
XLIX, 1086; Lade, Deutsch. med. Wchnschr., 1913, XXXIX, 693; Kallos, Ibid., 1885; 
Leschly and Boas, Hospitalstid, 1913, VI, 640; Thomsen and Boas, Ztschr. f., Immunitats- 
forsch., 1913, XVI, 430; Olitsky and Olmstead, Jour. x\m. Med. Assn., 1914, LXII, 293; 
Bing and Schmitz, Ugesk. f. Laeger, 1916, LXXVIII, 1119; Perutz, Wien. klin. Wchnschr., 
1916. XXIX, 1619; MacCann, Jour. Lab. and Clin. Med., 1919, IV, 742. 

- Deutsche med. Wchnschr., 1914, XL, 1607; Sem. Med., 1914, XXXIV, 361; Miinch. 
med. Wchnschr., 1914, LXI, 2093; see, also, Frankel and Thiele, Ibid., 2095; Cole and 
Chin, Arch. Int. Med., 1915, XVI, 880; Brandt, Deutsche med. Wchnschr., 1915, XLI, 915. 

3 Ann. de I'lnst. Pasteur, 1912, XXVI, 657 and 737; Ibid., 1913, XXVI, 341. 



720 DIAGNOSTIC METHODS 

In the performance of this test there are, so to speak, three phases, 
which may be outlined as follows: (i) Mix the heated serum with varying 
dilutions of the cytozyme (alcoholic extract of tissue, preferably human 
heart) and allow the mixtures to stand for 3^^ to i hour to permit the in- 
activation of the cytozyme by the serum; (2) add solution of calcium chlorid 
and the serozyme (fresh plasma or recalcified oxalate plasma) and allow to 
stand for fifteen minutes to permit the production of thrombin, providing, 
of course, that cytozyme is available, the amount of thrombin formed being 
in direct ratio to the amount of cytozyme present; (3) add oxalate plasma to 
test for the presence and amount of thrombin and time the reaction of 
coagulation. 

Reagents Necessary. 

1. Oxalate Plasma. — Into a 100 c.c. volumetric flask place 10 c.c. of a i 
per cent, solution of sodium oxalate to which has been added 3^^o volume of 
10 per cent, solution of sodium chlorid (this latter is added to render the solu- 
tion isotonic for the blood, according to the suggestion of Uemura^). With a 
short, stout, sterilized needle obtain the blood from the external jugular vein 
of a sheep or goat, collecting slightly more than 100 c.c. Allow a few c.c. of 
this blood to flow out of the syringe and add the remaining blood to the 
fluid in the flask, up to the 100 c.c. mark. Before drawing the blood it is 
wise to heat the solution in the flask to 4o°C. and moisten the interior 
of the flask with this warm oxalate solution to prevent the condensation of 
steam from the warm blood on the wall of the flask, as this may produce some 
hemolysis. During the addition of the blood to the solution in the flask, 
shake gently in order to mix thoroughly and prevent coagulation, as every- 
thing possible must be done to prevent the formation of thrombin at this 
stage. The oxalated blood is now poured into centrifuge tubes, which have 
been previously rinsed with warm physiologic salt solution, and centrifuged 
for 15 to 20 minutes; the supernatant fluid is then transferred to fresh cen- 
trifuge tubes and again centrifuged for 30 minutes at high speed to remove 
the platelets. The resulting plasma should be absolutely clear and free from 
hemoglobin; if colored at all, it should be nothing more than a yellow. This 
will keep in the ice-box for one to two weeks. When used in the test, this 
oxalate plasma is diluted as follows: i part oxalate plasma; 5 parts physio- 
logic salt solution; and Y2 part of i per cent, sodium oxalate solution. 
Should coagula appear in this plasma after standing for a few days, these 
may be filtered off through sterile cotton wool. 

2. The Serozyme. — This is prepared from the above oxalate plasma as 
follows: To 50 c.c. of this oxalate plasma add 5 c.c. (one-tenth volume) of 
I per cent, calcium chlorid solution; mix well and place in the incubator for 
at least 15 minutes. The plasma should have coagulated by this time, but, 
if it has not done so or has only partially completed the process, add a few 
more drops of the calcium chlorid solution. Plasmas, which have heen kept 
for some time, often will not coagulate at all, or, at least, very slowly; such 

1 Am. Jour. Med. Sc, 1917, CLIV, 533. 



THE BLOOD 721 

can not be used for the preparation of the serozyme but may be satisfactory 
where the oxalate-plasma, itself, is directed. When the coagulum has 
formed, grasp it with a long forceps and express the serozyme by pressing 
with a wringing movement. Should a second coagulum appear, this should 
be removed in the same manner. Freshly prepared serozyme may contain 
traces of thrombin, so should not be used for testing purposes until at least 
one or two hours have elapsed, in order to be certain that all tendency to 
coagulate is passed. When used in the test, this serozyme is diluted with 
five volumes of physiologic salt solution and employed in the proportion of 
0.5 c.c. per tube. Before use a prehminary test should show that it is satis- 
factory, that is that it contains neither cytozyme nor thrombin. For every 
test, it should be controlled in this direction, by placing 0.5 c.c. of the diluted 
serozyme in a tube with i c.c. of the calcium chlorid solution, without any 
cytozyme, and adding i c.c. of oxalate plasma. This mixture should not 
coagulate, at least not until the following day. According to Uemura, weak 
serozyme may be considerably improved by diluting it with 10 volumes of 
distilled water and allowing it to stand for several hours; to the cloudy 
mixture is added sufficient lo per cent, sodium chlorid to make it contain 
0.8 per cent. NaCl, after which it may be employed in doses of i c.c. 

3. Calcium Chlorid Solution. — ^This is prepared by mixing 5 c.c. of a i 
per cent, solution of calcium chlorid with 95 c.c. of physiological salt solution. 

4. The Cytozyme. — This is an alcoholic extract of tissues which contains, 
especially, the lecithin group of lipoids. These may be the same alcoholic 
extracts that are used as antigens in the Wassermann test, the extract of 
human, beef, or guinea-pig heart being apparently preferable. The con- 
centration and effectiveness of this extract should be such that, diluted 20 
to 40 times and tested as given below, c.i c.c. causes the oxalate plasma to 
coagulate in one to three minutes. To test its strength proceed as follows: 
In two test-tubes place o.i c.c. of 1:20 and 1:40 dilutions of this extract in 
physiologic salt solution respectively, add to each tube i c.c. of the calcium 
chlorid solution, 0.5 c.c. of the serozyme solution and i c.c. of the oxalate 
plasma dilution. Coagulation should result in i to 3 minutes. In the actual 
performance of the test, four dilutions are employed in constant dose of o.i 
c.c. : namely, i : 40; i : 80; i : 160; i : 320. The coagulation time will, of course, 
differ with each dilution. 

5. The Serum. — This is collected as for the Wassermann test. It must 
be clear and absolutely free of corpuscles and dissolved hemoglobin. Ue- 
mura has shown that admixture of small quantities of hemoglobin would 
affect the sera in such a way as to diminish their inhibition of coagulation. 
The serum resulting from spontaneous coagulation is very likely to contain 
cytozyme. For this reason, before use in the test, the sera should be heated 
for I hour, the optimum temperature being 56 to 58°C., as Toyama^ has 
shown. Specimens received through the mails may contain a relatively 
large amount of this cytozyme and may require heating to 60 for i hour, but 
the results obtained with such heated sera may show false positive reactions. 

^ Am. Jour. Syph., 1919, III, 6. 

46 




m 



722 DIAGNOSTIC METHODS 

Technic. 

Before beginning the actual test, it is assumed that the reagents have all 
been tested as to their activity. The test outlined above under Cytozyme, 
will show, of course, that these are active as far as the actual coagulation is 
concerned. If the coagulation does not result within three minutes, the 
trouble is usually due to defective serozyme or cytozyme. Further, the 
reagents should be tested against known positive and negative syphilitic 
sera, to show that the reagents, as prepared, do produce a delay in coagulation 
of the syphiHtic as compared with the normal serum. 

The test is conducted as follows: 

1. For each serum to be tested arrange 5 small, clean, sterile test-tubes 
in a row; into each of the first four place o.i c.c. of 1:40, 1:80, i: 160, and 1:320 
salt solution emulsions of the alcoholic extract of human heart (cytozyme), 
respectively. The fifth tube is the serum control and receives o.i c.c. of 
salt solution. 

2. To each of the 5 tubes add 0.1 c.c. of heated serum; mix well and 
stand aside at room temperature for an hour. 

3. To each tube add i c.c. of the calcium chlorid solution and 0.5 c.c. of 
the diluted serozyme solution; mix well and stand aside at room temperature 
for 15 minutes. 

4. To each tube add i c.c. of the diluted oxalate plasma, recording ac- 
curately with a watch the time when oxalate plasma was added; mix well 
and examine each tube every minute for coagulation. 

5. Controls. — The fifth tube of each series is the serum control and should 
remain fluid for several hours at least and, usually, until next day or even 
indefinitely. Should coagulation occur in this tube, a fresh specimen of the 
serum should be heated to 60 for an hour and the test repeated. Each dilu- 
tion of cytozyme in dose of 0.1 c.c. should be set up without serum (substi- 
tuting O.I c.c. of normal salt solution) and should show coagulation in from 
I to 6 minutes, the higher dilutions showing, of course, the longer period. 
Further, positive and negative syphilitic sera should be run through at the 
same time. 

An accurate record of time in minutes must be kept of each tube of the 
series and the serum controls, until the tubes of normal serum containing 
cytozyme and the tubes containing all reagents except serum show coagula- 
tion; after this each tube should be examined at close intervals. The serum 
controls should show no coagulation. Normal or nonsyphilitic sera usually 
coagulate within 10 minutes, but this interval may be shorter or long r (an 
hour or more) depending on the quality of the cytozyme, serozyme, and oxa- 
late plasma. Strongly syphilitic sera delay coagulation for considerably 
longer periods; weakly syphilitic sera delay coagulation only slightly beyond 
the time required for normal serum. No standard time can be laid down 
for the coagulation of normal or syphilitic serum, as this varies with each new 
lot of reagents. Coagulation is indicated by the development of a firm jelly- 
like coagulum in the tubes. The results are expressed, as given by Kol- 



THE BLOOD 723 

mer and Toyama,^ as ±, +, and + + . The first indicates slight delay in 
coagulation; the second a delay of 5 to lo minutes; and the third a longer 
delay. For no coagulation the fluid state may be indicated by fl. 
G. Tests before Transfusion. 

As transfusion of blood is becoming a matter of more or less frequent 
occurrence, it is advisable to test the blood for isoagglutinins and isohemolysins 
in every possible case. ''That donor should be chosen whose blood shows no 
inter-agglutination or hemolysis with the patient's serum and corpuscles. If 
such a donor cannot be obtained, it is safer to use a person whose serum is 
agglutinative toward the patient's cells than one whose cells are agglutinated 
by the patient's serum" (Kolmer). 
Technic. 

Two or three c.c. of blood are obtained from each donor by the usual sero- 
logical method. Place 0.5 c.c. at once in a centrifuge tube containing 5 c.c. 
of a I per cent, solution of sodium citrate in normal salt. Place the re- 
mainder of the blood in a small dry test-tube and allow the serum to separate. 
Obtain 3 or 4 c.c. of blood from the recipient in the same manner and follow 
the same procedure with this blood as with that of the donors. 

Centrifuge the sodium citrate tubes, pipet off the supernatant fluid and 
wash the cells again with normal salt solution. Repeat if necessary in order 
to free the cells from every trace of serum. After the final washing, add 
enough normal salt solution to the cells to make a total volume of 5 c.c. 

Centrifuge the serum tubes and separate the clear serum. This serum 
should show no trace of hemolysis. The following series of tests should then 
be set up. In this work the blood should not be allowed to stand for a 
period of time before the elements are separated, and the actual tests should 
be begun within 24 hours of the withdrawal of the blood. Use small sterile 
test-tubes about 8 by i cm. 

1. Four drops of donor's serum + i drop of recipient's red-cell emulsion. 

2. Four drops of recipient's serum + i drop of donor's red-cell emulsion. 

3. Control: Four drops of donor's serum -|- i drop of donor's red-cell 
emulsion. 

4. Control: Four drops of recipient's serum -{- i drop of recipient's red- 
cell emulsion. 

5. Control: One drop of donor's red-cell emulsion -}- 4 drops of normal 
salt solution. 

6. Control: One drop of recipient's red-cell emulsion -|- 4 drops of 
normal salt solution. 

One c.c of normal salt solution is added to each tube and the tubes are 
shaken and placed in the incubator for two hours, being inspected about 
every one-half hour. Agglutination in tubes i and 2 may be recognized 
macroscopically by the clumping of the red cells. Hemolysis is noted as in 
previous tests. If there is any question about hemolysis, let the tubes stand 
in the ice box over night and read them as in the complement-fixation tests. 
Tubes 3, 4, 5, and 6 should show no hemolysis or agglutination. 

1 Am.' Jour. Syph., 1918, II, 505. 




I 



724 DIAGNOSTIC METHODS 

Besides the above tests, it is advisable to test the donors with the Wasser- 
mann test. Further, immediate members of the patient's family offer the 
least risk in transfusion and should be obtained whenever possible. 

The above macroscopic method, while reliable, is, nevertheless, rather 
cumbersome and time consuming. For these reasons, Rous and Turner^ 
introduced a rapid and simple method, which involved the collection of the 
blood in a pipet holding 0.25 c.c. of fluid, the mixing of the recipient's blood 
and that of the donor in Wright's pipets, and the microscopic examination 
of the mixtures for agglutination, in a few minutes if there is an emergency, 
or at the end of fifteen minutes, if time is not a factor. With this technic, the 
difficulty not infrequently arises that clots may form and interfere with the 
test. Minot^ has brought out a modification of this test, which is more simple 
and does away with the collection in the pipet, thus obviating the difficulty 
with the original technic. This latter worker collects the specimens as fol- 
lows: Place 3 or 4 drops of a 1.5 per cent, sodium citrate solution in 0.9 per 
cent, physiological salt in a small test-tube. Collect, from a prick in the finger 
or ear, in this tube 9 drops of blood from the recipient and one from the 
donor and in another tube collect 9 drops from the donor and one from the 
recipient. Mix by shaking, allow to stand for 15 minutes, and examine 
microscopically for agglutination. This method yields as accurate results as 
those mentioned above and is much more easily carried out, where the recip- 
rocal study of recipient's and donor's blood is undertaken. 

Grouping of Bloods. 

Through the work of Landsteiner and others it has been established that 
human blood has two different isoagglutinins. These have been designated 
with the capital letters A and B. The elements, which are agglutinable are 
resident in the corpuscles and have been identified by the letters a and b. 
It is self evident that a blood can not contain either agglutinin A or B and 
its corresponding agglutinable substance a or b, as this would lead to aggluti- 
nation of the blood corpuscles by its own plasma. However, there are four 
different combinations of these elements, which are possible and which are 
shown to exist in the blood. This has lead to the study of the grouping of the 
bloods of individuals, based upon their agglutinin content as well as upon the 
power of their corpuscles to be agglutinated by other sera. While the early 
workers, as Landsteiner, Descatello and Sturli, and Hektoen, classified the 
blood in three groups, today we recognize 4 such groups and follow the classi- 
fication of Jansky or of Moss. These two latter systems of grouping the 
bloods differ merely in the placing of the most common group. Jansky 
styles this group I, while Moss classifies it as group IV. In other words, 
groups I and IV of Jansky's classification become Groups IV and I of the 
Moss grouping. Groups II and III of each classification remains the same. 
While the Jansky grouping has priority, yet it has not been so widely adopted 
as the Moss system. In this discussion we will follow the Moss grouping, 
inviting attention to the points mentioned above for those who prefer the 

1 Jour. A. M. A., 1915, LXIV, 1980. 

2 Boston Med. and Surg. Jour., 1916, CLXXIV, 667. 



THE BLOOD 



725 



Jansky method. The value of this study of grouping of blood is shown 
when blood is to be selected for transfusion. If the group to which a recipient 
belongs is known or determined, as outlined below, a proper blood may be 
selected from a list of donors, which may be available and which belong to 
the same group as the recipient. 

The four possibilities of combination of the agglutinating and agglu tin- 
able elements mentioned above are as follows: 

[ Plasma contains no agglutins. 

Corpuscles contain a and b. 

Plasma contains agglutinin A. 

Corpuscles contain b. 
I Plasma contains agglutinin B, 
• Corpuscles contain a. 



Group I. 



Group II. 




Plasma contains agglutinins A and B. 
Corpuscles contain no agglu tinable elements. 

From a study of the above groupings, it naturally follows that: 

{a) the 'plasma or serum of an individual of Group I will not agglutinate 
the corpuscles of any other group and that the corpuscles of this group are 
agglutinable by the sera of all other groups; 

{h) the plasma or serum of Group II will agglutinate the corpuscles of 
Groups I and III, while the corpuscles of this group are agglutinable by sera 
of Groups III and IV; 

(c) the plasma or serum of Group III will agglutinate the corpuscles of 
Groups I and II, while the corpuscles of this group are agglutinable by sera 
of II and IV. 

{d) the plasma or serum of Group IV will agglutinate the corpuscles of 
Groups I, II, and III, while the corpuscles of this group are not agglutinable 
by the sera of any of the other groups. 

The above points, which characterize the serum and corpuscles of each 
of the 4 blood groups, as far as their agglutinating actions is concerned, may 
be noted from the following graphic representation, which is taken from 
Sanford.^ In this figure, the corpuscles of the various groups are agglu- 
tinated by the sera of the groups from which the arrows head. 
I . Ill 




The proportional representation of these four groups among human 
individuals, as far as investigated cases indicate, is, according to Moss, as 
follows: Group I, 10 per cent.; Group II, 40 per cent.; Group III, 7 per cent.; 

1 Jour. A. M. A., 1916, LXVII, 808; Journal-Lancet, iqi7,XXXVII, 698; Jour. A. 
M. A., 1918, LXX, 1221. 



726 



DIAGNOSTIC METHODS 



Group IV, 43 per cent, 
largest group. 



In the Jansky classification, Group I represents the 



Methods of Determining Groups. 

In any method for such study one should have on hand either sera or 
corpuscles of Groups II and III, at least, as with these other specimens of 
sera or corpuscles may be grouped. While it is an advantage to have, also, 
the sera and corpuscles of Groups I and IV, yet these are not absolutely 
essential, as will be seen from the following considerations. The serum of 
Group II agglutinates the corpuscles of Groups I and III, while that of Group 
III agglutinates the corpuscles of Groups I and II. As the corpuscles of 
Group IV are non-agglutinable, all the groups are covered by testing the 
corpuscles of the unknown blood against the sera of Groups II and III. So, 
too, in cases in which the sera of the unknown blood is submitted for exam- 
ination, alLthat is necessary is to have on hand corpuscles of Groups II and 
III for the following reasons. Corpuscles of Group II are agglutinable by 
sera of Groups III and IV, while those of Group III are agglutinable by sera 
of Groups II and IV. As the sera of Group I will not agglutinate the cor- 
puscles of any of the other groups, all the four groups may be detected by 
such methods. 

Several methods have been introduced for grouping the bloods of pros- 
pective recipients and donors, the microscopic methods being the ones of 
choice. The test of Moss consists simply of mixing one drop of serum with 
one drop of an approximately i per cent, suspension of twice washed red cells 
on a cover glass and suspending over a hanging-drop slide, studying the prog- 
ress of agglutination under the microscope. Minot has found that the wash- 
ing of the cells, as advocated by Moss as well as by Rous and Turner in their 
work, is not necessary if one collects the blood in citrate solution. He, 
therefore, recommends the following method, which is followed in the writer's 
laboratory for such work. Collect i drop of the blood, from a prick of the 
finger or ear, in i c.c. of 1.5 per cent, sodium citrate solution in 0.9 per cent, 
salt solution. Then follow the Moss technic of mixing one drop of the un- 
known serum with one drop of this suspension of blood cells of both Groups 
II and III. Allow to stand for fifteen minutes at room temperature and 
examine under the microscope. It is unnecessary to use the hanging-drop, 
as the simple placing of the inverted cover glass on the ordinary glass slide 
will give entirely satisfactory results, especially if a film of petrolatum be 
placed around the edges of the slip. Brem has called attention to the fact 
that, in using the microscopic method, one must be on his guard lest the pres- 
ence of isohemolysins may interfere with these group determinations, owing 
to the possibility that these may break up the agglutinated corpuscles so 
rapidly that the agglutination may be masked. He uses two loopsful of 
serum mixed with i loopful of corpuscle suspension, instead of the drops as 
advocated by Minot. If one has on hand corpuscle suspensions of Groups II 
and III, instead of the sera of these groups, the method is the same, except 
that the sera becomes then the unknown element in the test. 



THE BLOOD 72 7 

Sanford has introduced a further modification of this method by ad- 
vocating the use of dried sera of Groups II and III, against which the cor- 
puscular suspensions of the unknown blood are tested. After determining 
the grouping of the blood of various individuals, two loopsful of the serum 
of Group II and, also, of Group III, are placed on separate cover slips and 
allowed to dry in the air. This method has the advantage that any number 
of such dried specimens may be prepared and saved for future work. As the 
isoagglutinins are thermostabile, no effect is manifest in the later tests. On 
receipt of the specimen of unknown blood, a corpuscular suspension is pre- 
pared as advocated by Minot, and i loopful of this placed on the dried serum. 
This is inverted over a glass slide, rimmed on the edges with petrolatum and 
studied microscopically. If the unknown corpuscular suspension is to be 
tested the results are as follows: agglutination on both slides of Groups II 
and III, indicates Group I; no agglutination. Group IV; agglutination of 
unknown by III serum. Group II; agglutination of unknown by II serum. 
Group III. If unknown serum is to be tested, add one or two loopsful of 
corpuscle suspension of known Groups II and III and proceed as above. 
The results are as follows: agglutination of corpuscles on both slides. Group 
IV; no agglutination. Group I; agglutination of Group II corpuscles, Group 
III; agglutination of Group III corpuscles, Group 11.^ 

IX. Medicolegal Aspects 

In medicolegal examinations one is frequently called upon to determine 
the identity of various suspected stains. Such examinations may tax the 
entire resources of the worker and have, in many cases, led to absolutely 
negative results. It is not sufficient at the present day that the examiner 
state that a certain stain is blood, but he must be prepared to say what kind 
of blood it is. This latter point has been made possible by recent work so 
that one is fairly sure of limiting this statement at least to the blood of very 
closely associated animals. 

(i) Red Cells. 

It is not always a simple matter to determine that a stain on cloth, wood, 

iron, etc., is really due to blood. The color of the stain may be of any hue 

from dull red to a dirty gray, depending upon its exposure to various elemental 

conditions. The action of various substances, such as mortar, brick, or lime 

^ See Landsteiner, Wien. klin. Wchnschr., 1901, XIV, 1132; Descatello and Sturli, 
Miinch. med. Wchnschr., 1902, XLIX, 1090; Hektoen, Jour. Infect. Dis., 1907, IV, 297; 
Jansky, Shorn, klin. y. Praze, 1907, VIII, 85; von Dungern and Hirschfeld, Miinch. med. 
Wchnschr., 1910, LVII, 741; Ztschr. f. Immunitatsf., 1910, VIII, 526; Moss, Bull. Johns 
Hopk. Hosp., 1910, XXI, 63; Am. Jour. Med. Sc, 1914, CXLVII, 698; Ottenberg, Jour. 
Exper. Med., 1911, XIII, 425; Weil, Jour. A. M. A., i9i5,LXIV, 425; Ottenberg and Lib- 
man, Am. Jour. Med. Sc, 1915, CL, 36; Lewisohn, Ibid., 886; Simons, Jour. A. M. A., 
1915, LXV, 1339; Satterlee and Hooker, Ibid., 1916, LXVI, 618; Lindeman, Ibid., 624; 
Cherry and Langrock, Ibid., 626; Brem, Ibid., LXVII, 190, Unger, Ibid., 1917, 
LXIX, 2159; Williamson, Jour. Lab. and Clin. Med., 1917, II, 58; Wohl, Ibid., 516; 
Turner, Journal-Lancet 191 7, XXXVII, 230; Meleney, Stearns, Fortuine, and Ferry, 
Am. Jour. Med. Sc, 1917, CLIV, 733; Hedon, Presse Med., 1917, XXV, 129; Coca, Jour. 
Immunol., 1918, III, 93; Lyon, Jour. A. M. A., 1919, LXXII, 1134; Drinker and Britting- 
ham. Arch. Int. Med., 1919, XXIII, 133; Lewisohn, Presse Med.. 1919, XXVII, 593; 
Moffitt, Klugh and Shepard, Jour. A. M. A., 1919, LXXIII. 1821 



728 DIAGNOSTIC METHODS 

in any form, strong acids and alkalies, leather, chemicals in wall-paper, 
starched clothing, etc., may so change the blood or its reactivity to certain 
tests that no definite conclusions can be obtained. 

It is self-evident that the demonstration under the microscope of blood- 
cells is the surest proof of the presence of blood, the size and shape of the 
corpuscles frequently giving a clew as to the source of the blood. It becomes, 
therefore, necessary to make a suspension of the stained material preferably 
in isotonic (0.9 per cent.) sodium chlorid solution. The degree of solubility 
of the stain will depend upon the age of the stain, the heat to which it has 
been subjected, the amount of sunlight or of moisture to which it has been 
exposed, and the material upon which the stain is formed. Various fluids 
have been advanced, from time to time, as dissolving agents for the stains, 
many of these are active laking or hemolytic agents, especially for dried 
red cells, so that one sees in such preparations only the shadow of the red 
cell. Such laking agents are distilled water, 0.85 per cent, ammonium chlorid 
solution, 50 per cent, glycerin solution, and many others. Frequently a 
strong 30 per cent, potassium hydrate solution will enable the worker to 
obtain a very good idea of the presence or absence of red cells. Marx's 
fluid (made as follows, hydrochlorate of quinin 10 c.c. of a i to 1,000 solution 
added to 10 c.c. of 33 per cent, potassium hydrate solution, and tinted with 
eosin which will stain the erythrocytes a characteristic reddish hue) is a very 
excellent examining fluid. 

As the presence of the red cells is so hard to demonstrate, search for them 
is frequently omitted and reliance placed more upon the demonstration of 
the hemoglobin in such stains. If the stain is upon a hard surface it may be 
scraped off with a clean piece of glass. If it be upon cloth, a portion of the 
stained part and also of the unstained part is removed and cut into small 
pieces with scissors which are absolutely clean. Such small pieces not broader 
than I mm. are very easily handled and can be readily teased out if desired.^ 

(2) Guaiac Test. 

This test originally devised by van Deen is one of our oldest for the detec- 
tion of blood coloring matter, but seems to be much more reliable in its nega- 
tive phase than when positive. The principle of the test is as follows: To a 
watery solution of the suspected stain is added an equal portion of fresh tinc- 
ture of guaiac. This tincture is best made by dissolving the guaiac resin in al- 
cohol when needed. The addition of the guaiac tincture to the suspected 
solution causes a milky turbidity. On now adding ozonized oil of turpentine, 
peroxid of hydrogen, or oil of eucalyptus, so that these latter substances float 
upon the guaiac and blood solution, a distinct blue color will be manifested at 
the point of contact, and, on shaking the tube, the coloration will spread 

^ Blood stains on cloth may be removed, even after years standing by treating the stains 
with a drop of acetic acid and then soaking for several hours in a concentrated (70 per cent.) 
aqueous solution of chloral hydrate. Also, treatment with hydrogen peroxid solution for 
several hours will remove the stain. While these methods are reliable for the removal of 
blood stains, they are not to be used for forensic purposes as the tests to be applied for recog- 
nition of blood are interfered with to a great extent, although Boas employs the above chlor- 
al hydrate solution in his improved test for occult blood in feces (see Berl., klin. Wchnschr., 
1916, LIII, 1357)- 



THE BLOOD 729 

throughout the mixture. This test seems to be very delicate, demonstrating 
the presence of blood in a dilution of several thousand. The blue coloration 
observed in this test is due to the oxidation of the guaiac to guaiaconic acid, 
which is then further oxidized into guaiac blue by the catalytic action of the 
oil of turpentine. It has been shown by Taylor that this reaction is also given 
by many substances among which we find manganate and permanganate of 
potassium, peroxid of manganese, peroxid of lead, chlorin, bromin, iodin, 
nitric and chromic acids, ferric chlorid, salts of copper, ferro- and ferricyanid 
of potassium, gum acacia, gluten, unboiled milk, raw potato pulp, pus, and 
any living cell or its intracellular enzymes. Various enzymes such as the 
oxidases will give this reaction. Buckmaster advises the use of the pure 
guaiaconic acid along with peroxid of hydrogen in the place of the guaiac 
resin and believes that the test increases in delicacy and accuracy under these 
conditions. He states that fluids containing hemoglobin or most of its de- 
rivatives give this test when it is impossible to detect pigments by any other 
methods and rightly adds that boiling the fluid suspected of containing 
blood does not interfere with the reaction but, on the contrary, throws out of 
consideration the action of milk, pus, fibrin, or of any enzyme. For the 
proper performance of this test the fluid to be tested, should not be alkaline 
and only very slightly, preferably not at all, acid. 

(3) Schaer's Test. 

This test is similar in many ways to the van Deen test, but employs, instead 
of guaiac, a i to 4 per cent, solution of aloin in alcohol. On adding this tincture 
to the suspected solution a red color soon becoming distinctly cherry-red appears 
upon the addition of ozonized oil of turpentine. Many other substances give 
a pink color, but only after the lapse of one or two hours, while the color 
with blood appears in a very short time. The tincture of aloin should always 
be freshly prepared, as of itself it undergoes this color change after standing. 

(4) Phenolphthalin Test. 

This test,^ originally employed by Meyer ^ and, later, by Utz,^ has been so 
improved by Kastle and Amoss^ and Delearde and Benoit^ that it is, probably, 
our most delicate one for the detection of blood, especially in stains, its limit 
of delicacy being about i part of blood in 8 million of water. As in the pre- 
vious tests, the hemoglobin acts as an oxygen carrier, the active oxidizing 
agents being ozonized turpentine or hydrogen peroxid. The oxidases, peroxi- 
dases and catalases play small roles in this process in the case of blood-stains, 
but, in the various secretions and excretions of the body, their influence is 
very great. 

Phenolphthalin is a product of the reduction of phenolphthalein by zinc in 
alkaline solution. When oxidized in alkaline solution, it is converted into 
phenolphthalein with production of an intense red color. The reagent may 

1 See Kastle, Bull. 51, Hyg. Lab. U. S. P. H. & M. H. S., 1909; Boas (Berl. klin. Wchn.- 
schr., 1916, LIII, 1357) advises the use of thymolphthalein. 

2 Miinch. med. Wchnschr., 1903, L, 1489. 

3 Chem. Ztg., 1903, XXVII, 11 51. 

4 Bull. 31, Hyg. Lab. U. S. P. H. & M. H. S., 1906. 

^ Comp. Rend. Soc. de biol., 1908, LXIV, 990. See Kelly, Jour. Lab. and Clin, Med., 
1916, I, 897. 



1 



1 



11 



730 DIAGNOSTIC METHODS 

be obtained in the market or, preferably, prepared as follows. Phenolphtha- 
lein is dissolved in a considerable excess of 30 per cent, sodium hydrate solu- 
tion and boiled with an excess of zinc dust until a few drops of the strongly 
alkaline liquid no longer give a red color after neutralization with HCl and 
sufficient alkali to alkalinize the solution. The solution is then decanted from 
the excess of zinc dust and the phenolphthalin is precipitated by acidifying 
with HCl. Collect the precipitate on a filter and purify by repeated crystalli- 
zation from water and alcohol. This purification is continued until a white 
crystalline compound is obtained free from every trace of phenolphthalein 
(as shown by absence of red coloration on addition of alkali). Dry at room 
temperature or in the oven at 50° to 8o°C., care being taken to avoid contact 




Fig. 157. — Hcemin Crystals from Human Blood. {Hawk.) 

Reproduced from a micro-photograph furnished b}^ Prof. E. T. Reichert, of the 

Universit}' of Pennsylvania. 

with metallic surfaces. This compound should be kept in tightly stoppered 
bottles in a dark place, as oxidation gradually occurs. Rarely do we find 
that the coloration is sufficient to interfere with the tests, but in the delicate 
forensic tests it may be necessary to repurify a product which has stood for 
some time.^ 

The solution, as used in the test, is as follows: Mix a slight excess of 
phenolphthalin, prepared as above, with i c.c. of N/io sodium hydrate solu- 
tion and a few c.c. of redistilled (from glass) water, shake thoroughly and 
filter. To the filtrate add 20 c.c. of N/io NaOH solution, o.i c.c. of 3 per 
cent, hydrogen peroxid solution and make the mixture up to 100 c.c. This 
solution shows no trace of pink coloration when fresh, but gradually acquires 

^ For routine laboratory work this reagent may be prepared as follows: Two grarns of 
phenolphthalein, 20 grams of sodium hydrate, 10 grams of zinc dust and 100 c.c. of distilled 
water are mixed and heated until the solution becomes colorless. Filter while hot and pre- 
serve in an amber bottle. This solution is prone to show a slight reddish tinge from time to 
time owing to reoxidation, so that it may be necessary to heat it with zinc dust again and 
refilter. 



THE BLOOD 73 I 

a color, which may become so intense that the reagent cannot be employed. 
In forensic work, use only freshly prepared solutions. 

To I part of the aqueous solution of the stain or of the secretion or excre- 
tion to be tested add 2 parts of the reagent and allow to stand for a few min- 
utes. In the presence of blood a pink to red color appears, the intensity de- 
pending on the amount of blood present. This reaction is retarded by the 
extracts of various animal tissues or various secretions of the body. For this 
reason we are never able to detect as small amounts of blood in the secretions 
as in watery solutions of pure blood, the limit of delicacy of all reactions being 
far less in the. former than in the latter case. Boiling the solutions before 
applying the test removes most of the interfering factors. If the secretion be 
treated with a thick cream of albuminium hydrate suspension, the precipitate 
will carry down the blood pigment and thus concentrate it. A small amount 
of this precipitate, whether derived from saliva, urine, feces suspension, milk, 
or exudates, will show a decided red color when added to 2 c.c. of the reagent. 
Of course, in applying the test to the aqueous solution of the blood stain, no 
such treatment need be employed. 

This test is especially recommended for the detection of blood in all secre- 
tions and excretions (boiling them before applying the test) and, particularly, 
in the examination of suspected stains for forensic purposes. All possible 
fallacies must be guarded against and confirmatory tests used if necessary.^ 

(5) Teichmann's Test. 

This is one of the most important tests for the presence of blood and 
when positive is conclusive proof of the kind of stain with which one is work- 
ing. A drop of blood or a portion of the suspected stain is spread upon a glass, 
slide and covered with a drop of a very dilute solution (o.oi per cent.) of com- 
mon salt. 2 The salt solution is then evaporated at a low temperature. A few 
drops of glacial acetic acid are then placed upon the salted stain and the 
preparation is covered with a cover-glass. The acid is now slowly evaporated 
by holding the slide over a flame in such a way that the fluid steams but does 
not boil. As the acid evaporates, more is allowed to run under the cover- 
glass, this addition and evaporation being done thrice. The specimen is 
cooled and mounted in glycerin or distilled water, after which it maybe exam- 
ined with the dry lens. A successful specimen, which is not always obtained, 
w^l show the presence of numerous brownish rhomboid crystals, which are 
separate or grouped in sheafs or rosettes. These are the hemin crystals or 
hydrochlorate of hematin, called, after their discoverer, Teichmann^s crystals. 

It very frequently happens that the specimens are not successful, so that 

1 Vas (Deutsch. med. Wchnschr., 191 2, XXXVIII, 141 2) calls attention to the possi- 
bility of the presence of phenolphthalein as a purgative (if feces be tested). The addition of 
alkali alone will give a red color under this condition. See Cade and Mulsaut, Lyon Med., 
191 2, CXIX, 885; also de Jager, Berl. klin. Wchnschr., 1914, LI, 795; Schirokauer, Deutsch. 
med. Wchnschr., 1914, XL, 1472 and 161 7; Schneider and von Teubern, Ibid., 1673; Boas, 
Ibid., 1915, XLI, 549. 

2 Nippe (Deutsch. med. Wchnschr., 1912, XXXVIII, 2222) advises one to mix the speci- 
men with a few drops of a solution of o.i gram each of potassium bromid, iodid and chlorid 
in 100 grams of glacial acetic acid. This modification shows up the crystals even when 
blood is mixed with rust. See, also, Symons, Biochem. Jour., 1913, VII, 596. 



W 732 DIAGNOSTIC METHODS 



I 

I 



several slides should be made before a negative report is given. Reasons for 
failure may be found in the excessive heat applied to evaporate the acid, in 
the fact that the salt solution may have been too concentrated, or that the 
stain may have been very old. The addition of salt seems to be unnecessary 
except in the case of old blood stains or when the blood is poor in salts. It 
should even then be added in only very small amounts. As pointed out by 
Rose, when blood is mixed with iron rust the hemin test is usually negative.^ 

(6) Spectroscopic Examination of Blood. 

Certain characteristic appearances of the blood are noted on spectroscopic 
examination. 2 The spectrum of the various blood pigments has been given in 
previous sections so that only a few additional remarks are necessary in this 
place. In spectroscopic examinations, the use of the ordinary hand spectro- 
scope is all that is necessary, the more complicated ones adding little to the 
differentiation of the various spectra. If the blood be fresh and the hemo- 
globin unaltered the spectrum is, of course, that of oxyhemoglobin; but as the 
usual stains submitted for examination are frequently very old ones, such a 
spectrum is practically never seen. The end-product of the alteration of 
hemoglobin as found in old blood stains is hematoporphyrin, which is iron- 
free and can be identified easiest by the spectroscope. The suspected stain is 
dissolved in concentrated sulphuric acid yielding a reddish-violet fluid, which 
is then examined spectroscopically. The spectrum of hematoporphyrin has 
been given previously. Many blood stains can be recognized only by this 
test, so that it is always advisable to make a spectroscopic examination, espe- 
cially if other tests for hemoglobin have failed.^ 

(7) Precipitin Test. 

The precipitin test has been discussed in great detail previously (page 
677). This is the absolute identification test for a stain, providing the stain 
has been shown to be a blood stain by the tests given above. The precipitin 
test is specific, within very narrow limits, and is recognized in law as reliable in 
differentiating human blood from that of the domestic animals. Its one fal- 
lacy is that it reacts with the blood of anthropoid apes just as it does with that 
of man, but in most cases this element may be easily excluded. 

X. Value and Limitations of Blood Examinations 

It has been truly said that the value of a blood examination ''is measured 
by the practical use which may be made of it, and not by any interesting yet 
useless information it may throw on the case." In clinical work we are prone 
to follow well-established lines of procedure and to forget, when engaged in 
counting the cells in an obscure case, that " the meaning and aim of the clini- 
cal study of the blood covers a much wider field than is embraced in the mere 

^ See Symons, Biochem. Jour., 1913, VII, 596; Beam and Freak, Ibid., 1915, IX, 161; 
Bokarius, Vierteljahrschr. f. gerichtl. Med., 1918, LV, 255. 

2 Lewis reports a study of the ultraviolet absorption spectra of blood sera (Proc. Roy. 
Soc. London, 1916, LXXXIX, 327). 

3 See Sutherland and Mitra, Biochem. Jour., 1914, VIII, 128; Heller, Vierteljahrschr. 
f. ger. Med.. 1916, LI, 219 calls attention, to the importance of fluorescence of hemoglobin 
derivatixes in forensic blood tests. 



THE BLOOD 733 

investigation of the histological elements." Yet our knowledge regarding 
the variations in the plasma is so meager that the busy practitioner may be 
pardoned for such neglect.^ 

There can be little question that the so-called "blood diseases" have 
the most characteristic variation from the normal, yet even here it is not so 
easy, as one might assume, to make an indisputable diagnosis in all cases. 
Thus, aside from the pure parasitic diseases, leukemia and advanced pernici- 
ous anemia seem to be the only ones capable of a certain diagnosis from the 
examination of the blood. True the various anemias may be detected, but 
the etiology is not always clear nor the differentiation of a primary from a 
secondary form invariably possible. 

In recent years the study of the chemistry of the blood has assumed 
great importance. There can be little question that carefully controlled 
chemical examinations of the blood do give information which can be ob- 
tained in no other way. The importance of a determination of the amount 
of the various nitrogenous constituents and the relation of these amounts to 
those excreted in the urine is self evident when attempts are made to make 
careful diagnoses and prognoses of conditions associated with an acidosis. 
While these determinations require a certain experience in chemical technic, 
yet the different methods advised are soon mastered by close study, so that 
one may avail himself of the benefits offered by routine use of such methods 
in properly selected cases. Further, a study of the blood sugar will, often- 
times, make a differential diagnosis between a true diabetes from one of the 
"renal" type as well as from a glycosuria, induced by purely alimentary 
excess of carbohydrate. A study of these methods is strongly recommended. 

It is, of course, evident that bacteriologic examination of the blood and 
the application of the agglutination reaction may clear up many an obscure 
case, but such work requires much more time and much more attention to 
detail than is at the disposal of the busy practitioner. It is, however, in just 
this class of infectious fevers that much benefit, in a confirmatory w^ay, is 
forthcoming from a careful study of the blood. Thus, a leucocytosis or a 
leucopenia in any suspected case might lead to a differentiation of central 
pneumonia from typhoid fever or a scarlet fever from a case of measles. 
Moreover, a leucocytosis follow^ing a leucopenia in typhoid would strongly 
indicate perforation and consequent surgical interference. Suppurative proc- 
esses anyw^here in the system are usually associated, unless very limited in 
extent, with a polynuclear neutrophile leucocytosis; so that the leucocyte 
count may be of great importance in judging of the extension of a pus infec- 
tion. Immediate operation is occasionally decided upon after a leucocyte 
count, but this should not be made the only basis for such intervention unless 
frequent counts have given the surgeon the definite knowledge that a leuco- 
cytosis has suddenly occurred. 

It should be stated as an axiom that blood findings may never be inter- 
preted except in the light of the clinical findings. To the clinician it should 
be said, never trust the laboratory report implicitly unless it agrees with the 

^ See Butterfield and Stillman, Am. Jour. Med. Sc, 191 7, CLIV, 783. 



w 



734 



DIAGNOSTIC METHODS 



clinical manifestations; to the laboratory worker we would say, never report 
a blood finding as diagnostic without knowing something of the clinical his- 
tory of the case. 

A single blood examination will rarely be of any greater value than will 
a single temperature determination. It is the series that enables one to 
decide either as to diagnosis or as to operative or therapeutic procedures. 
What we need most is not so much blood work, but much better work when 
done. Too much reliance is often placed on blood reports, so that a word of 
caution does not seem ill advised. Never rely solely on the blood findings, 
but use them merely as one of many links in your chain of clinical evidence. 
In this way mistaken diagnoses will occur less frequently. 



BIBLIOGRAPHY 



Jena, 1904. Diagnose 



1. Arneth. Die neutrophilen weissen Blutkorperchen. 

und Therapie der Anamien. Wiirzburg, 1907. 

2. Bang. Methoden zur Mikro-bestimming einiger Blutbestandteile. Wiesbaden, 

1916. 

3. Besancon et Labbe. Traite d'hematologie. Paris, 1904. 

4. Bramwell. Anemia and Diseases of the Ductless Glands. Edinbtirgh, 1899. 

5. BucKMASTER. The Morphology of Normal and Pathological Blood. Phila- 

delphia, 1906. 

6. Cabot. Clinical Examination of the Blood. New York, 1904. 

7. Canon. Bakteriologie des Blutes. Jena, 1905. 

8. Celli. Malaria According to the New Researches. London, 1900. 

9. Champy. Le sang et les maladies du sang. Paris, 19 13. 
ID. Charles. Fonction des leucocytes. Paris, 1904. 

11. Coles. The Blood: How to Examine It. London, 1898. 

12. Craig. Estivo-autumnal Malarial Fevers. New York, 1901. The Malarial 

Fevers. New York, 1909. 

13. Da Costa. Clinical Hematology. Philadelphia, 1905. 

14. Decastello and Krjukoff. Untersuchungen iiber die Struktur der Blut- 

zellen, Wien, 191 1. 

15. Ehrlich, Lazarus, Pinkus, und von Noorden. Blut-Krankheiten. Wien, 

1913. Translation, Philadelphia, 1905. 
r6. Ehrlich und Lazarus. Die Anamie. 3te Auf . Wien, 19 13. 

17. EwiNG. Clinical Pathology of the Blood. Philadelphia, 1903. 

18. Gilbert et Weinberg. Traite du sang. Paris, 1913. 

19. Gradwohl and Blaivas. The Newer Methods of Blood and Urine Chemistry. 

St. Louis, 191 8. 
20., Grassi. Die Malaria. Jena, 1901. 

21. Grawitz. Klinische Pathologic des Blutes. Leipzig, 191 1. 

22. Hayem. Du sang. Paris, 1889. 

23. Helly. Die haemopoetischen Organe. Wien, 1906. 

24. Janeway. The Clinical Study of Blood-pressure. New York, 1904. 

25. Kahane. Die Chlorose. BerHn, 1901. 

26. Kaplan, Serology of Nervous and Mental Diseases. Philadelphia, 1914. 

27. Kolmer. Serology. Philadelphia, 1914. 

28. Laveran. Du paludisme et de son hematozoaire. Paris, 1891. Traite du 

Paludisme. Paris, 1907. 

29. Laveran et Mesnil. Trypanosomes and Trypanosomiases. Chicago, 1907. 

(Translation by Nabarro.) 

30. Lenhardt. L'Anemia a type chlorotique. Paris, 1906. 



THE BLOOD 735 

31. VON Limbeck. Klinische Pathologic des Blutes. Jena, 1896. 

32. Mannaberg. Die Malariakrankheit. Wien, 1899. 

33. Naegeli. Blutkrankheiten und Blutdiagnostik. Leipzig, 191 1. 

34. Oliver. Studies in Blood-pressure. London, 1908. 

35. Pappenheim. Atlas der menschlichen Blutzellen. Jena, 1905 and 1912. 

Clinical Examination of the Blood and Its Technique. New York, 19 14. 

36. Patella. I leucociti non granulosi del sangue. Torino, 1906. La genesi 

endoteliale del leucociti mononucleali del sangue. Siena, 1907. 

37. Potain. La Pression arterielle de rhomme. Paris, 1902. 

38. Prowazek. Handbuch der pathogenen Protozoan. 2te Aufl. Leipzig, 1914- 

15- 

39. Reinert. Die Zahlung der Blutkorperchen. Leipzig, 1891. 

40. RuGE. Malaria Krankheit. Jena, 1906. 

41. Schleip. Atlas der Blutkrankheiten. Berlin, 1908. Translation, New York, 

1908. 

42. ScHOENBORN. Gcfrierpunkts- und Leitfahigkeitsbestimmungen. Wiesbaden, 

1904. 

43. Stephens and Christophers. The Study of Malaria. London, 1905. 

44. Sternberg. Primarerkrankungen. Wiesbaden, 1905. 

45. Strauss und Rohnstein. Anamien. Berhn, 1901. 

46. Sutherland. Blood Stains. New York, 1907. 

47. Tallqvist. Ueber experimentelle Blutgiftanamien. Helsingfors, 1899. 

48. Thayer. Lectures on the Malarial Fevers. New York, 1897. 

49. Thayer AND. Hewetson. Malarial Fevers of Baltimore. Baltimore, 1895. 

50. Thomas and Ivy. Applied Immunology. Philadelphia, 1915. 

51. Turk. Vorlesungen ueber klinische Haematologie. Wien, 1904 and 19 12. 

52. Uhlenhuth. Das biologische Verfahren zur Erkennung und Unterscheidung 

von Menschen und Tierblut. Jena, 1905. 

53. Ward. Bedside Hematology. Philadelphia, 1914. 

54. Watkins. Diagnosis by means of the Blood. New York, 1902. 

55. Weiss. Haematologische Untersuchungen. Wien, 1896. 

56. Wile. Blood Examination in Surgical Diagnosis. New York, 1908. 

57. Wolff. Die Kernzahl der Neutrophil en. Heidelberg, 1906. 



)4 



CHAPTER IX 
TRANSUDATES AND EXUDATES 

I. General Considerations 

The serous membranes are normally kept moistened by liquids whose 
quantity is only sufficient in a few instances, as in the pericardial cavity and 
the subarachnoidal space, for a complete chemical analysis to be made of 
them. Under pathological conditions an abundant transudation may take 
place from the blood into the serous cavities, into the subcutaneous tissues or 
under the epidermis. If such conditions be the result of circulatory dis- 
turbance, the kidneys are usually unable to eliminate the normal amount of 
fluid from the system and, as a result, the retained fluid collects both in the 
serous cavities and in the areolar tissue. Such accumulations of fluid, 
known as transudates, are similar to the lymph, being, as a rule, poor in cellular 
elements and yielding little or no fibrin. These transudates must be sharply 
differentiated from the accumulations of fluid, which are the result of direct 
l. inflammatory processes in the membranes lining the serous cavities and are 

known as exudates. These latter fluids are generally rich in cellular elements 
and yield relatively more fibrin. As a rule, the richer a transudation is in 
leucocytes, the closer it stands to pus, while the poorer it is in these elements 
the closer it resembles true lymph. 

The formation of true transudates is largely a question of filtration under 
the influence of the rate of blood flow, the blood pressure, the irritation of 
the capillary endothelium, and the variable permeability of the endothelial 
cells. We should expect, therefore, that the passage of dissolved substances 
from the blood would be regulated by the same laws that control the secretion 
of physiologic fluids, namely the laws of passage of fluids through semiper- 
meable membranes. The crystalloids would be, therefore, in approximately 
the same concentration as in the blood plasma, while the colloids must be in 
far less concentration, the actual values being influenced, of course, by the 
special membrane through which the fluid passes. The condition of the blood 
would hence affect the chemical composition of such transudates, hydremic 
plasma yielding a fluid poorer in solids, while anhydremic blood is associated 
with a transudate of higher specific gravity. 

From a clinical standpoint a differentiation between transudates and 
exudates is not infrequently impossible, so that it is advisable to resort to 
aspiration of the fluid and to chemical and microscopical examination of the 
material withdrawn. The chief phases of such examinations are: (i) the 
chemical and physical properties of the fluid; (2) the bacteriological aspect 
of the fluids, and (3) the morphological characteristics of the cellular elements. 

736 



TRANSUDATES AND EXUDATES 737 

Obtaining the Specimen. 

Whenever fluid is to be withdrawn, either for diagnostic or therapeutic 
purposes, it is necessary to resort to puncture of the cavity containing the 
fluid. In all cases the site of puncture must be as carefully prepared as in 
any surgical procedure. Puncture may be performed with the ordinary 
trocar or, preferably, with a large needle which has a rather large lumen. The 
instruments must be carefully sterilized before use. If the skin is especially 
tough, it is advisable to make a small incision through the skin and insert the 
needle through the incision. Very little pain is felt by the patient during 
this procedure, but the writer is accustomed invariably to resort to the use of 
ethyl chlorid to anesthetize the part. 

In the withdrawal of pleuritic effusions the spot selected should be neither 
too high nor too low. It may be in the seventh intercostal space in the 
axillary line or in the eighth intercostal space at the outer angle of the scapula. 
The arm of the patient should be brought forward with the hand resting on 
the opposite shoulder, in order to widen the intercostal spaces. In inserting 
the needle it is wise to make the thrust close to the upper margin of the rib 
so as to avoid the intercostal artery. In all cases the fluid should be with- 
drawn slowly and the excess above that required for examination allowed to 
drain until the desired amount is obtained. If the puncture is for diagnostic 
purposes, lo to 20 c.c. are sufficient, while the therapeutic withdrawal of 
fluid will vary with the amount present and with the clinical symptoms of the 
case. Should the patient show signs of shock or of depression during the 
operation, the procedure should be interrupted as quickly as possible. 
Aspiration is rarely necessary or advisable. 

In the withdrawal of fluids from the abdominal cavity, the needle or 
trocar is thrust through the lower abdominal wall and the fluid collected. In 
this procedure more or less danger of puncturing the bowel exists if the 
effusion be small, so that the needle should not be carelessly inserted lest this 
complication arise. Naturally, in well-marked ascites no such danger is 
present. 

II. Physical and Chemical Properties 

Transudates are, as a rule, serous in character, usually transparent, 
colorless, or light yellow in color, but at times showing a milky, reddish, or a 
greenish tinge, the latter practically always being observed after the fluid has 
stood exposed to the air. Such solutions are, as a rule, dichroic, yellow by 
transmitted light and green by reflected light. They are alkaline in reaction 
and show a specific gravity, which varies, according to the origin of the fluid, 
from 1,006 to 1,018, while serous exudates from the same cavities show a 
much higher specific gravity.^ The variations in specific gravity depend 
largely upon the amount of albumin present in the transudate, this practically 
never being over 3 per cent, and usually much lower. The chief proteins 
present are albumin and globulin, these being related to one another in the 
transudates as one and one-half to one, while in the exudates the globulin 

^ See Trevisan, Ztschr. f. exper. Path. u. Therap., 191 2, X, 141. 

47 



738 DIAGNOSTIC METHODS 

is relatively much increased.^ The determination of the total protein may 
be made by methods of fractional precipitation as previously discussed. 
The transudates from the pleura contain the largest percentage of albumin, 
while edematous fluids rarely show over i per cent. Transudates do not 
coagulate spontaneously. Glucose is present both in transudates and exu- 
dates in amounts varying between 0.04 and o.i per cent. The mineral 
constituents of transudates are somewhat higher than in the exudates, the 
former averaging 0.96, the latter 0.89 per cent. Under pathological in- 
fluences fat, blood, uric acid, and biliary pigment may find their way into 
both types of fluid. In diabetes an excess of sugar and the presence of 
acetone bodies may be detected. 

The exudates are usually straw- or lemon-yellow in color depending on 
the degree of inflammation, or they may assume colorations ranging from a 
deep red (hemorrhagic) to a milky (purulent) shade. Biliary pigments may 
cause a bright green, while various medicaments, such as methylene blue, may 
produce a greenish-blue coloration. The specific gravity is almost invariably 
above 1,018, the reaction is alkaline, the albumin content is usually above 
3 per cent., reaching as high as 7 per cent., while the globulin is relatively 
much increased in comparison with the albumin. This globulin increase is 
largely due to paraeuglobulin. Nucleoprotein is especially abundant in 
purulent exudates in which the autolytic processes are more or less marked. 
The total nitrogen of the various fluids varies from 0.22 to 1.38 per cent. The 
nitrogen partition may be seen from the following table of Gerhartz.^ Exu- 
dates coagulate spontaneously on standing.^ 



Figures in terms 

of % (grams per 

100 c.c.) 



Total 1 ffui?^' I Protein ' Ammonia | Purin Urea I Amino-acid 

N I ^^j5^^ n ; N N N I n 



Transudate |0. 22-0. 58 

Serous exudate 0. 43-1 . 09 

Purulent exudate. . .,1.1 i-i . 33 



. 18-0 . 53 o . 1 7-0 .520. 007-0 .010. 002-0 . 007 .01-0 .050. 002-0 . oos 
0.39-1 .oy'o. 37-1 03 o. 01 -0.03 o.oi o.oi-o.o6i 0.007 

0.94-1.22 I. 14 0.01 0.007 0.02-0.1 0.004-0.28 



The exudates, which, accurately speaking, are always of inflammatory 
origin, may be serous, serofibrinous, seropurulent, hemorrhagic, purulent, 
putrid, chylous, and chyloid. 
Serous Exudates. 

These are clear, of a light straw color, and show a specific gravity above 

1,018. There is a large amount of fibrin, as shown by the dense network 

^ See Mosny, Javal and Dumont, Presse med., 1914, XXII, 477; Ujihara, Biochem. 
Ztschr., 1914, LXI, 55; Berl. klin. Wchnschr., 1914, LI, 1112; Villaret, Jour, de Physiol, et 
de Path, gen., 1913, XV, 617, 652 and 875. 

2 Chemie der Transudate and Exsudate, Jena, 1908, See, also, Wiener (Biochem. 
Ztschr., 191 2, XLI, 149) regarding the presence of proteolytic ferments and amino-acids in 
exudates; and Lenk and Pollak, Deutsch. Arch. f. khn. Med., 1913, CIX, 351. See Hegler 
and Schumm, Med. Klin. ,'1913, IX, 1810; Dorner, Deutsch. Arch. f. khn. Med., 1914, CXIII, 
342; Denis and Minot, Arch. Int. Med., 1917, XX, 879; Schulman, Jour. A. M. A., 1017, 
LXVIII, 1256, reports a case of pleural effusion containing a large percentage (9.73) of 
cholesterol. 

3 Rivalta advocates the following test to differentiate an exudate from a transudate: 
In a conical glass place about 200 c.c. of water and add 2 drops of glacial acetic acid. Mix 
thoroughly and allow i or 2 drops of the puncture-fluid to fall into this weak acid solu- 
tion. If the fluid be an exudate a distinct cloud will be observed in the wake of the falling 
drop, while if it be a transudate little or no turbidity mil be noticed. This reaction is 
due to the large globulin content of the exudate. 



TRANSUDATES AND EXUDATES 739 

microscopically, containing a few red cells which may be derived from the 
bleeding at the point of puncture, a few leucocytes which may vary in type 
according to the kind of bacteria causing the infection, and large endothelial 
cells from the serous membrane lining the cavity. If the blood-cells be pres- 
ent in sufficient numbers to give a distinct red color to the fluid it is termed a 
hemorrhagic exudate, while if a few pus-cells are found it may be called a 
seropurulent type. The gradations between the true serous and serofibrin- 
ous types of exudate are exceedingly varied, so that it is difficult to tell which 
type is really present. Even from a purely serous exudate a certain amount 
of coagulation, with formation of a distinct fibrin network, may be obtained, 
so that the only criterion w^ould be one of degree.^ 

The type of leucocyte present is usually of the polymorphonuclear variety, 
although other forms ma}^ be present. It is, therefore, an important part 
of the examination of an exudate to determine the percentage relations of these 
various cellular elements. This will be discussed in the section on Cytology. 
Chylous Exudates. 

On account of the close relationship between the abdominal, thoracic, 
and pericardial cavities, on the one hand, and the large lymphatic trunks, on 
the other, it is possible for lymph to pass directly into these cavities in case 
rupture of these vessels occurs. Such exudates show all the properties of 
chyle. The fluid is white and milky, contains between 1.5 and 2.5 per cent, 
of protein, and a considerable amount of fat, which may be demonstrated by 
staining with osmic acid or Sudan-Ill or by alkalinizing with sodium hydrate 
and shaking out with ether. 
Chyloid Exudates. 

It has been found that carcinoma, tuberculosis, extreme cardiovascular 
changes, hepatic disturbances, puerperal sepsis, and infection with the filaria 
may give rise to a chyloid type of ascites. The fluid in these cases is less 
milky than that of the true chylous type, contains less fat, and is not so com- 
pletely cleared by shaking w4th ether. It contains serum-albumin, mucin and, 
especially, rather large amounts of a complex of pseudoglobulin and lecithin, 
to which the opacity is due.^ Traces of sugar are found but not as large in 
amount as in the true chylous types. These fluids resist putrefaction for 
long periods owing to their lecithin content. The'accumulation of fluid after 
tapping is much more rapid in the chylous than in the pseudochylous types. 
Hemorrhagic Exudates. 

This type is, in reality, a serofibrinous form containing large numbers 
of blood-cells. It is observed in patients with hemorrhagic diathesis, in 
connection with active tuberculosis, with neoplasms of the serous cavities, 
and following injuries to the chest or abdomen. In this form of exudate, the 
exciting bacterial agent, usually the tubercle bacillus, may occasionally be 
found, but not always. The type of leucocyte (mononuclear) would be very 

^ See Epstein, Jour. Exper. Med., 1914, XX, 334. 

2 See Wallis and Scholberg, Quart. Jour. Med., 1910, III, 301; Ibid., 191I: IV, 153; also 
Henry, New York Med. Jour., 191 1, XCIV, i; and Lecliard, Brit. Jour. Child. Dis., 1913, 
X. 433; Fubini, Gazz. d. osp., 1915, XXXVI, 145; Outland and Clendening, Jour. A. M. A., 
1916, LXVI, 1833; Lewin, Am. Jour. Med. Sc. 1916, CLII, 71. 



I 
k 

W 740 DIAGNOSTIC METHODS 

strong presumptive evidence in favor of tuberculosis, even though no bacilli 
were found. If the exudate be due to a malignant growth, it is sometimes 
possible to obtain shreds of the tumor tissue and thus make a probable 
diagnosis. In judging of the malignancy of the cells, it is sometimes difficult 
to differentiate the abnormal from the normal, especially in the affections of 
the pleura. These malignant cells are usually extremely large and are char- 
acterized by their vacuolation and fatty degeneration (see cut). It is not 
infrequent to find in hemorrhagic exudates, which have remained in the body 
cavity for some time, a large number of cholesterin crystals and occasionally 
small masses of hemosiderin. 

Purulent Exudates. 

These are composed either of true pus or of seropus. They are more or 
less yellow in color, thick and occasionally tenacious, and separate on standing 
or centrifuging into a cellular deposit and a pus serum. The cells forming 
the pus are not infrequently in a condition of advanced fatty degeneration 
and may contain numerous bacteria. The addition of acetic acid will usually 
clear up the cells so that the nuclei become recognizable. Fat is present in 
amounts as high as 7 per cent., while a relatively large amount of various pro- 
teins and extractives is shown on chemical examination. Fatty acid crystals 
and cholesterin may be abundant in the sediment, especially in old abscesses. 
Fresh pus is usually alkaline, but may become distinctly acid, owing to the 
development of lactic acid in the process of autolysis.^ 

Purulent exudates are investigated with special regard to the type of cell 
present and to the organism associated with the pus formation. In ordinary 
pus, the cell is of the polymorphonuclear type, although occasionally the 
mononuclear form may be predominant. The bacteria are very numerous 
and include many of the most important types. While the pyogenic bacteria 
are more frequently the various types of staphylococci and streptococci, it is 
to be remembered that other organisms may produce pus under certain condi- 
tions. Thus the typhoid bacilli, colon bacilH, pneumococci, Friedlander's 
bacilli, gonococci, diphtheria bacilli, Morax-Axenfeld and Koch- Weeks bacilli, 
and influenza bacilli, among others, may be the causative factor in the forma- 
tion of a purulent exudate. It will be seen, therefore, that the bacterial ex- 
amination of a purulent exudate may require a very extensive research. As it 
is frequently impossible to decide, from a stained specimen, as to the special or- 
ganism causing the infection, cultures should be made in every doubtful case. 
The peculiarities of such cultures may be found in any work on bacteriology. 

Putrid Exudates. 

These may be observed in various cavities of the body or in the substance 
of various organs, especially the liver and lungs. They arise from the 
entrance of pus into these cavities from perforation of a gangrenous area, 
gastric or intestinal ulcer, malignant growths, etc. The material obtained by 
puncture is usually brownish or greenish in color, has a very offensive odor, 
and is usually alkaline, but may be acid. Microscopically degenerated cells, 

1 See Ito, Jour. Biol. Chem., 1916, XXVI, 173. 



TRANSUDATES AND EXUDATES 741 

numerous bacteria, cholesterin, fatty acid, and hematoidin crystals are ob- 
served. In some cases various portions of an echinococcus cyst may be found 
in the exudates. Bilirubin crystals and various amino-acids may be found 
in rupture of an hepatic abscess. 

III. Bacteriology 

It is always advisable to make cultures of the material obtained by punc- 
ture in order to discover the organism which is acting as the exciting cause 
of the condition under investigation. Usually a few c.c. of the fluid are 
allowed to drop into a flask containing 50 c.c. of sterile nutrient bouillon and 
the mixture incubated for 24 to 48 hours. From the growth, obtained in this 
preHminary work, subcultures are made on various media and microscopical 
examination employed to identify the organism. It is always advisable to 
make at least two microscopical specimens, staining one with the ordinary 
methylene blue stain and the second with Gram's stain. 

Tubercle Bacilli. 

If these organisms are suspected, the technic of staining is the same 
as outlined under Sputum. It frequently happens, in the examination of 
suspected tubercular exudates, that the presence of a large amount of fibrin, 
either with or without spontaneous coagulation of the specimen, makes it 
exceedingly difficult to find the organism, even though it be present. In 
such cases, advantage is taken of a procedure, recommended by Jousset,^ 
known as inoscopy. If the fluid has not coagulated, it is allowed to do so 
in order that the coagulation may enclose the bacilli within the fibrinous 
network. The coagulum is separated as completely as possible from the 
supernatant fluid, is washed with distilled water, and is treated with 30 to 
40 c.c. of the following mixture, which will digest the fibrin. 

Pepsin, 2 grams. 

Sodium fluorid, 3 grams. 

Glycerin, 10 c.c. 

Hydrochloric acid (cone), 10 c.c. 

Distilled water, q.s,, ad., 1000 c.c. 

This mixture is placed in the incubator for 24 hours, when the fluid becomes 
homogeneous. The digested fluid is centrifuged and smears are made from 
the sediment as previously described. It is always advisable to treat such 
smears with a small quantity of albumin fixative, so that the organisms may 
not be washed from the slide. The smear is fixed in the flame and stained in 
the usual way with carbol fuchsin. The tubercle bacilli may not appear as 
deeply stained after such treatment. This method has been replaced, to a 
large extent, by the Antiformin method (see p. 18). 

Gonococcus. 

The gonococcus appears in stained specimens as small biscuit-shaped or 
coffee-berry-shaped cocci, which are arranged in pairs separated by a narrow 
^ Semaine Med., 1903, XXIII, 22. 




^^ 742 DIAGNOSTIC METHODS 

unstained portion. Occasionally two of these pairs of hemispheres are joined 
together, forming tetrads. They may be stained by any of the anilin dyes, 
but are recognized especially by their reaction toward Gram's method. In 
this connection it must be said that other organisms, among which we find the 
diplococcus intracellularis meningitidis of Weichselbaum and the micrococcus 
catarrhalis, resemble the gonococcus both in morphological and staining char- 
acteristics, so that a differentiation by this method is not always possible. 
Fortunately, however, the cultural peculiarities of these organisms absolutely 
. differentiate them.^ 

Gram's Stain. 

The principle of the staining of various organisms by Gram's method 
is that certain organisms retain the primary color after treatment with de- 
colorizing agents, while others lose this primary stain and must be treated 
with a contrast stain for their recognition. If the organism retains the 
primary blue color it is called Gram-positive, while if it lose the primary color 
and take on the contrast stain it is called Gram-negative. Among the organ- 
isms which are Gram-negative we find the gonococcus, meningococcus, micro- 
coccus catarrhalis, influenza bacillus, typhoid bacillus, colon bacillus, Koch- 
Weeks bacillus, and the Morax-Axenfeld bacillus; while the tubercle bacillus, 
smegma bacillus, diphtheria bacillus, pneumococcus, streptococcus, staphylo- 
coccus, and various saprophytic cocci found in the smears both of the male 
and female urethra are Gram-positive. 

The solutions required in Gram's method are: (i) an anilin oil — gentian 
violet mixture, consisting of 84 c.c. of anilin water (water saturated with 
anilin and filtered), and 16 c.c. of a saturated alcoholic solution of gentian 
violet; (2) a solution of iodin consisting of one gram of iodin, 2 grams of potas- 
sium iodid, dissolved in 300 c.c. of water; (3) a dilute solution of carbol 
fuchsin, a dilute aqueous solution of safranin, or a i per cent, aqueous solution 
of Bismarck brown as a contrast stain. Jensen^ has shown that 0.5 per cent, 
aqueous solution of methyl violet (6B) may be used as the primary stain and, 
thus, obviate the necessity of preparing fresh anilin water solutions, as 
applied in the original Gram method. This worker advocates, also, the use 
of a somewhat stronger iodin solution, namely, iodine i gram, potassium 
iodid, 2 grams, dissolved in 100 c.c. of water. 

Technic. 

Smears of the exudate are made, in the manner previously described for 
making blood smears, by receiving a drop of the purulent material upon 

^ See Warden (Jour. Infect. Dis., 1913, XII, 93), who emphasizes the importance of a 
slightly acid reaction in the media containing preferably human body fluids, such as ascitic 
or hydrocele fluid. Warden (Jour. Infect. Dis., 1913, XIII, 124) calls attention to the fact 
that, oftentimes, what appears to be the gonococcus in smear preparations is shown to be 
a staphylococcus on culture. This awaits confirmation. See, also, Whitney, Boston 
Med. and Surg. Jour,, 1914, CLXX, 749; France, New York Med. Jour., 1914, C, 1255; 
Wolbarst, Ibid., 1915, CI, 146; Warden, Jour. Infect. Dis., 1915, XVI, 426; Pearce, Jour. 
Exp. Med., 1915, XXI, 289; Eastman, 111. Med. Jour., 1916, XXX, 396; Culver, Jour. A. 
M. A., 1916, LXVI, 553; Ibid., 1917, LXVIII, 362; Warden, Ibid., 432; Herbst, Ibid., 761- 

2 Berl. klin. Wchnschr., 1912, XLIX, 1663; see, also, Special Report No. 19, Med. Res. 
Comm., National Health Insurance, London, 1918, p. 6; Stovall and Nichols, Jour. A. M. A., 
1916, LXVI, 1620. 



PLATE XXXIII. 



^y 



^^ 



•^*J - ^ 



'"^^^ "^ 'f 



^w 






Xathar.n^ Hill- 



GoNOCocci IN Urethral Discharge, (Gram's Stain.) 



4 



TRANSUDATES AND EXUDATES 743 

one end of a glass slide and spreading it in a thin even layer by means of a 
second slide. These smears are then fixed by passing several times through 
the flame. When the slides have cooled, they are covered with the gentian- 
violet solution which is allowed to act for one to three minutes. The solution 
is then poured off and the excess removed by washing in water. Without 
drying, the iodin solution is placed on the slide and allowed to act for one- 
half to one minute. It is then washed in water and the preparation treated 
with 95 per cent, alcohol until no more color is removed by it. The alcohol 
is then removed by washing with water and the smear is covered with one of 
the contrast stains above mentioned, in the writer's laboratory the safranin 
solution, which is allowed to act for only a few seconds. The stain is then 
washed off with water and the slide dried between folds of filter-paper. 

The specimen under the microscope shows the Gram-positive organisms 
stained deep blue, while the Gram-negative bacteria and the bodies of the 
pus cells take the red safranin stain. In such specimens the gonococcus, 
if present, will be observed both intracellularly and extracellularly, the former 
being the more characteristic. The ordinary pus cocci may likewise be 
intracellular, but these are distinctly Gram-positive, while the gonococcus 
is Gram-negative. 

In the purulent exudate from the urethra, large masses of mucoid material 
may be present, which are known as gonorrheal threads. These may be 
found in the urinary sediment and are usually easily recognized. They con- 
tain masses of pus cells, within which may be found numerous gonococci. 
These shreds may persist for years in anyone with a history of a previous 
gonorrhea, but they may then contain no organisms. The cytology of gonor- 
rheal pus presents nothing characteristic beyond the presence of numerous 
eosinophiles with large numbers of polynuclear neutrophiles and an occasional 
lymphocyte. 

It is to be stated that the gonococci need not be intracellular, as all or 
almost all of them may be extracellular in the very acute stages of the dis- 
ease and, also, in some cases of the chronic type. Further, it is to be remem- 
bered that some of the gram-positive organisms, when taken up by the 
leucocyte, may lose their staining characteristics and become gram-nega- 
tive. Also, in certain chronic cases, gram-negative organisms, such as the 
gonococcus, may hold the primary stain so tenaciously that examination 
shows these organisms to be gram-positive, although cultures indicate them 
to be true gram-negative gonococci. Such results are, fortunately, rare, 
and are due to the improper preparation of the slides. 

Smegma Bacilli. 

In the exudate of the preputial follicles, known as smegma preputii, are 
found fat globules, ammonium soaps, cholesterin crystals, and a few epithelial 
cells. In this smegma are found many bacilH, smegma bacilli, which show 
the same morphological and practically the same staining characteristics 
as the tubercle bacillus. This is differentiated, as" described under Sputum. 
If the urine is to be examined for tubercle bacilli, it is much better technic 



i4 



I 



744 DIAGNOSTIC METHODS 

to use a catheterized specimen than it is to resort to methods of questionable 
differentiation. If this is done any acid and alcohol-fast baciUi may be regarded 
as the tubercle bacilli, but in all cases the smegma bacilH must be excluded.^ 

Ducrey's Bacillus. 

This bacillus is the organism causing soft chancre and is known as the 
bacillus ulceris cancrosi. It is found in the purulent discharge from the 
chancre, but rarely, if ever, in pure culture. The sections of tissue may, 
however, show none but this organism. 

In making preparations for study of this organism, the ulcerated surface 
is scraped with a platinum loop, the pus spread upon a slide, dried in the 
air, and fixed with alcohol-ether or over the flame. It stains readily with 
all bacterial stains, but decolorizes with Gram's stain. 

In stained specimens the organism appears as a short, thick, oval bacillus 
with rounded ends and two lateral indentations, which occasionally give it 
the appearance of a figure 8. Ordinarily the extremities are more deeply 
stained than the central portion, this appearing almost clear. The organism 
has a tendency to form chains or groups, which are rarely found in the 
pus cells but are frequently seen within the epithelial cells. ^ 

Spirochseta Pallida. 

The Treponema PalHdum, discovered by Schaudinn and Hoffmann,^ is 
now definitely established as the causative factor of syphilis. It may be ob- 
tained from the primary chancre, the incised papules, condylomata, mucous 
patches, inguinal glands, as well as from the internal organs in tertiary and 
congenital syphilis. Its size, form, type of motility and method of cultivation 
have been discussed under blood. Noguchi^ states that there are many 
varieties of spirochete found in or about the body cavities, alimentary tract 
and genitalia and that these are nonpathogenic. He cites 8 species of Spiro- 
nema and 5 of Treponema as follows: Spironema refringens, S. microgyratum, 
S. buccalis, S; acuminatum, S. obtusum, S. pseudopallidum, S. eugyratum, 
S. stenogyratum and Treponema macrodentium, T. medium, T. microdent- 
ium, T. dentium, and T. calligyrum. Of these he shows that Spironema 
refringens, Treponema calligyrum and T. minutum (formerly regarded as 
identical with T. microdentium) are constantly observed in the male and 
female genitalia. 

^ See Brereton and Smith, Am. Jour. Med. Sc, 1914, CXLVIII, 267. 

2 In 1904 Scherber and Miiller (Arch. f. Dermat. u. Syph., 1904, LXXVII, 77) isolated 
and identified in cases of "erosive and gangrenous balanitis" (the so-called "fourth venereal 
disease") a fusiform bacillus and a spirochete, which appears to be similar, if not identical, 
with the organism of Vincent, discussed previously as the cause of certain types of stom- 
atitis. This organism has been given the name Spironema (spirochete) balanitidis and is 
rather thick as compared to the pallida and possesses very active motility, the motion being 
wave-like and rotary. It is about 0.2 microns in width and from 5 to 15 microns in length. 
There are usually from 6 to 10 spirals and are not as regular as those of the treponema 
pallidum. See, Corbus and Harris, Jour. A. M. A., 1909, LII, 1474; Corbus, Ibid., 1913^ 
LX, 1769; Noguchi, Jour. Lab. and Clin. Med., 1917, II, 383; Owen and Martin, Ibid., 
862. 

^ Arbeiten aus der kais. Gesundheitsamte, 1905, XXII, 527. 

^ Jour. Lab. and Clin. Med., 1917, II, 365 and 472; Jour. Exper. Med., 1918, XXVII, 
667; Ibid., XXVIII, 559. 



TRANSUDATES AND EXUDATES 745 

To obtain this organism from the primary lesions, the chancre is thor- 
oughly cleansed either with normal salt solution or with soap and water, 
rinsed with salt solution and dried. If the sore is healed, the epithelial cover- 
ing is removed. The chancre is lightly curetted or the edges scraped, the 
blood, which should be avoided, is wiped away and the serum which exudes 
used for the later work. A drop of this serum is placed upon a slide, covered 
with a cover-glass and examined at once under the dark-field illuminator, the 
spirochete appearing as colorless, glistening, rapidly motile spirals. If this 
method, which is to be preferred to any staining method, is not possible, a 
thin smear is made from the serum, transferring a drop by means of a 
platinum loop to a slide and using a rapid circular motion in spreading. The 
specimen is dried in the air and stained as follows.^ 

Giemsa's Stain. 

The air-dried film is fixed with chemically pure methyl alcohol and stained 
with Giemsa's stain (p. 538) for 20 hours, using i drop of the stain to i c.c. of 
water. The spirochete are stained a violet-red color, the ref ringens being blue. 

Ghoreyeb's Method. 2 

The smears are made as above and air-dried. Cover the smear with i 
per cent, aqueous solution of osmic acid for 30 seconds. Wash thoroughly 
in running water. Cover with solution of lead subacetate (Hquor plumbi 
subacetatis diluted 100 times) for 10 seconds. Wash in water. Cover with 
10 per cent, aqueous solution of sodium sulphid for 10 seconds. Wash in 
water. The above process is repeated three times. Following this the 
osmic acid solution is applied for 30 seconds, the specimen is washed in water, 
dried and mounted in balsam. The spirochete, bacteria and cellular detritus 
are stained black . This method is quick, reliable, and is to be recommended. 

India Ink Method. 

Burri^ has advanced this method, which is simple and rehable, although 
some specimens of ink may show confusing artefacts according to Barach,^ 
who used an inferior ink. A drop of the fresh serum from the lesion is placed 
at one end of a glass slide and immediately mixed with a small drop of 
Gun ther- Wagner India ink (Chin-Chin liquid pearl ink). The mixture is 
then spread and allowed to dry. The specimen is studied with the immersion 
lens. The whole field is a homogeneous brown or black color, the treponema, 
blood cells, etc., appearing as colorless highly refractile bodies. The 
morphology, length and numbers of spirals should distinguish the pallida 
from the other types. ^ 

^ Tunnicliff (Jour. Am. Med. Assn., 191 2, LVIII, 1682) calls attention to a very simple 
and reliable method formerly used by Oppenheimer and Sachs as well as by Ploeger. Make 
smears as usual, j&x in the flame and cover for a few seconds with a 10 per cent, mixture of a 
saturated alcoholic gentian-violet solution in 5 per cent, phenol. Wash in water and dry. 

2 Jour. Am. Med. Assn., 1910, LIV, 1498. 

^ Das Tuschverfahren, Jena, 1909. See, also, Walters, Jour. Am. Med. Assn., 1914, 
LXIIl, 1666. 

* Jour. Am. Med. Assn., 1910, LV, 1892. 

^ Goosmann (Jour. Cutan. Dis., 191 1, XXIX, 628) has advised the use of nigrosin as a 
substitute for India Ink. 



1 



l!H 



I 

Pi 



746 DIAGNOSTIC METHODS 

Tribondeau's Method. 

This^ is a modified and improved Fontana method and yields excellent 
results. It may be recommended for all routine staining of smears for the 
treponema, as it gives consistent and reliable microscopic pictures. 

Solutions Necessary. 

1. A fixing solution, consisting of i c.c. glacial acetic acid, 2 c.c. formalin, 
and distilled water 100 c.c. 

2. A mordant consisting of i gram of tannin dissolved in 20 c.c. of dis- 
tilled water. A small piece of camphor may be added to the solution to 
prevent contamination with molds. 

3. The staining solution (Fontana's solution): Silver nitrate, i gram dis- 
solved in 20 c.c. of distilled water. After solution is complete ammonia is 
added, drop by drop, until the precipitate first formed begins to dissolve and 
a faint opalescence remains. 

Technic. 

Films are made in the usual way, and dried in the air. Fix these films 
in the formol-acetic acid solution, which is poured on and off once or twice, 
a fresh quantity being left to act for i to 5 minutes. This procedure dis- 
solves out the hemoglobin, which may be present in the blood corpuscles in 
the film. In the case of secretion of syphihtic ulcers, it is wise to wash the 
films with absolute alcohol before applying the fixative. Smears from 
organs should have the fat removed by alcohol, ether, and finally alcohol. 
After treatment with the fixing solution, cover the preparation with the 
tannin mordant and heat gently until vapor rises (do not boil). Allow the 
mordant to act for 30 seconds. Wash thoroughly with tap water for 30 
seconds and follow with distilled water. Now pour on the silver solution 
and allow it to act for a few seconds in the cold. Pour this off and add a 
fresh quantity of the stain and heat gently until vapor arises. Allow the 
stain to act for a further 15 seconds, when the film is of a maroon color. 
Wash in distilled water for a few seconds and dry with blotting paper. The 
spirochetes are stained yellowish brown or blackish. The stained films may 
be examined directly in cedar oil, but this should not be left on as the oil 
destroys the preparation. The film may be mounted, if desired, in Canada 
balsam. 

Levaditi and Manouelian's Method. 

This method, 2 also known as the pyridin method, is applied only to the de- 
tection of treponema in tissues. It is superior to the older method of Levaditi. 
Cut the tissue into pieces about i to 2 mm. square, fix in 10 per cent, formalin 
for 24 hours and harden in absolute alcohol for 24 hours. Wash in distilled 

^ Bull. Soc. franc, de dermat. et Syph., 1912, XXIII, 474; Ann. de L'Inst. Pasteur, 
1917, XXXI, 425. See Fontana, Dermat. Wchnschr., 1912, LV, 1003. 

2 Compt. rend. Soc. de biol., 1906, LX, 134. See, also, Sobernheim, Kolle and Wasser- 
mann's Handbuch d. pathog. Mikroorg., 1913, VII, 770 



PLATE XXXIV. 






'■4ri 



ft> c i 



Katharihe >\\V\ 



Spirochete. Pallida in Tissue. (Levaditi's Stain.) 



1 



>i 



ii»<« 



TRANSUDATES AND EXUDATES 747 

water until the pieces fall to the bottom of the vessel. Place the tissue in a 
ground-glass stoppered bottle and add 50 c.c. of the following mixture. 

I per cent, aqueous solution of silver nitrate, 90 c.c. 
Pyridin, 10 c.c. 

Leave them in the silver solution for two to three hours at room temperature 
and for three to five hours at 5o°C. Wash in 10 per cent, pyridin solution. 
Immerse for three to four hours at room temperature in a freshly prepared 
solution with following formula: 

4 per cent, aqueous solution of pyrogallol, 90 c.c. 

Pure acetone, 10 c.c. 

Pyridin, ' 15 c.c. 

Remove from the reducing bath, dehydrate in absolute alcohol, clear in xylol 
and embed in paraffin. Cut sections and stain on the sHde with a 2 per cent, 
aqueous solution of toluidine blue. Differentiate in absolute alcohol or 
Unna's ether-glycerin mixture, dehydrate in absolute alcohol, oil of bergamot 
and xylol. Mount in balsam and examine. The spirochaete are stained a 
beautiful black, while the ground-work of the tissue is blue. 

Noguchi's Method. 

This method^ is used with beautiful results, especially with brain or cord 
tissue. Harden the tissue in 10 per cent, formalin for several days. The 
longer this is continued the better the results. Place the pieces (5 to 7 mm. 
thick) in the following mixture for five days at room temperature. 

Formalin, 10 c.c. 

Pyridin, . 10 c.c. 

Acetone, 25 c.c. 

Alcohol (absolute), 25 c.c. 

Distilled water, 30 c.c. 

Wash thoroughly in distilled water for 24 hours. Transfer to 96 per cent, 
alcohol for three days and, then, wash in distilled water for 24 hours. Place 
the tissue in an amber bottle and treat with 1.5 per cent, aqueous solution of 
silver nitrate for three days at 37°C. or five days at room temperature. Wash 
in distilled water for several hours. Reduce in the following mixture for 24 
hours. 

4 per cent, aqueous solution of pyrogallol, 95 c.c. 

Formalin, 5 c.c. 

Wash in distilled water, transfer to 80 per cent, alcohol for 24 hours, then to 
95 per cent, alcohol for three days and absolute alcohol for two days. Clear 
in xylol, xylol-paraffin and embed in paraffin. Cut sections from various 
strata of the tissue. Sections 3 to 5 /x give the best results. The trepone- 
mata are pure black in color, while the tissue varies from a pale yellow to a 
yellowish-brown. The neuroglia fibers sometimes stain distinctly, but are 
brownish, never black. 

1 Jour. Cutan. Dis., 1913, XXXI, 547. 



i 



748 



DIAGNOSTIC METHODS 



IV. Cytology 



The cytology^ of transudates and exudates has reference to the study 
of the various types of cells found in such fluids. As a rule, such investiga- 
tions, known as cytodiagnosis, are carried out more frequently on the non- 
purulent types of fluid, as the examinations of the purulent fluids are more 
especially concerned with the bacteria present. 

Technic. 

The technic of obtaining the cellular components of the puncture fluid 
will vary according as the effusion does or does not contain fibrin. If this be 
present in fairly large amount, the fluid must first be defibrinated before the 
next steps are possible. Here, again, the procedure may be complicated by 
the presence or absence of coagula. If the fluid be not coagulated, it is placed 
in a large sterile flask which contains a few sterile glass beads, the mixture 
is actively shaken until a firm clot is obtained, in the meshes of which a few 
of the cells will necessarily be enclosed, but the majority will remain sus- 
pended in the liquid. The fluid is then separated from the clot and placed 
in centrifugal tubes which are drawn out to a rather fine point. If the fluid 
be coagulated when collected, it should be shaken with glass beads to break 
up the clot and liberate the cellular elements. The fluid is then separated 
and treated as follows. 

The remaining portion of the technic is the same for fluids which contain 
or do not contain fibrinous material. The centrifuge is rotated rather 
rapidly for about ten minutes in order to collect the cells as a sediment. In 
most of the exudates the number of these cells is very small so that a concen- 
tration is absolutely essential. As Widal and Ravaut have show, the polynu- 
clear cells seem to be somewhat more affected by the defibrinization than the 
other types, so that these cells may show a relative diminution. After the 
cells have collected, the fluid is removed by rapidly inverting the tube in such 
a way that the sediment does not follow the liquid. Some workers advise 
the withdrawal of the sediment by a pipet with a long fine tip, but the writer 
has not found this method any more advantageous than the one spoken of 
above. The sediment in the tube is shaken so as to mix it thoroughly and a 
drop placed upon the glass slide and spread as described under Blood. Where 
very few cells are present it is wise to allow the drop to dry on the slide without 
spreading, in order to concentrate the cells in a small area. The specimen is 
then treated with Wright's stain and examined under the immersion lens. If 
stains are to be used which contain no fixative, such as methyl alcohol, it is 
necessary that the specimen be fixed by methods previously discussed in the 
section on Blood. The stain to be used will depend largely upon the points 
to be studied, the eosin-hematoxylin stain being especially serviceable in 
differentiating the nuclear structures of the cell. As the cells in the various 
pathologic fluids frequently show more or less degeneration, the nuclear por- 
tion is more suitable for study than is the cell protoplasm. For this reason 

^ See Labbe, Cytodiagnostic, Paris, 1903; Ravaut, Cytodiagnostic, Paris, 1901; Brion, 
Centralbl. f. allg. Path., 1903, XIV, 609. 



TRANSUDATES AND EXUDATES 749 

the writer prefers the use of the eosin-hematoxylin method along with the 
Wright stain or the triacid stain for the granules of the cells. 

These specimens may also be used for the study of the bacteria present. 
When this object is to be subserved, it is advisable to make two specimens, 
staining one with the ordinary methylene blue stain and a second by Gram's 
stain. If the material be very limited in amount, it is possible to combine the 
above staining methods by treating first with the eosin-hematoxylin method, 
washing in water and then following the ordinary procedure of the Gram 
method of staining. Such specimens are extremely panoptic and are espe- 
cially to be recommended. 

Cytology of Normal Fluids. 

The number of cellular elements in fluids from the various serous cavities 
of the body may vary from a very few to a large number. The cells observed 
are the red and white corpuscles of the blood, the latter of which are usually 
relatively more numerous than in the circulating blood and are usually largely 
of the polynuclear type, although mononuclear forms are frequently present. 
Neutrophiles and eosinophiles are present under normal conditions, the latter 
being relatively more abundant than in the blood. If a large number of red 
cells are found, an injury of the small vessels during puncture usually explains 
their presence. Besides these types of cell, which are exactly similar to those 
of the blood, a few endothelial cells are practically always seen. These cells 
are observed of different shapes, may be single or grouped in sheets, and may 
be very much degenerated. They are larger than the other cellular elements, 
their contour is usually circular, but may be irregular; they are mononuclear, 
and frequently contain round vacuoles. 

In examining normal as well as pathologic fluids for their cellular content, 
I GO cells should be counted if possible and the percentage of each type thus 
determined. This constitutes the cytologic formula of the exudate.. 

Cytology of Pathologic Fluids. 

According to the theory of Metchnikoff, the presence of a bacterial in- 
fection is associated with attraction of the leucocytes to the infected area. 
These cells then enter into combat with the bacteria and either destroy the 
organism or are destroyed by them. As has been shown, certain organisms, 
especially the tubercle bacillus and probably the typhoid organism, attract 
the lymphocytes, while most of the other organisms attract the polymor- 
phonuclear neutrophiles. Theoretically, therefore, it should be possible to 
decide as to a tubercular or nontubercular condition by the presence or ab- 
sence of an increased num.ber of the mononuclear types of leucocytes. This 
is the basis of the attempt at differential diagnosis by means of cytodiagnosis. 
Not infrequently one finds in malignant conditions the so-called specific 
cells which are either sarcomatous or carcinomatous. These, although 
specific, are not easy absolutely to identify. These cells are very large, fre- 
quently showing fatty degeneration, extensively vacuolated, and showing a 
mitotic mulberry-like nucleus. Although differing from the endothelial cell, 



75° DIAGNOSTIC METHODS 

confusion is very apt to arise, so that it is difficult to make a diagnosis in all 
cases from the appearance of such cells. 

Pleural Exudates. 
Primary Tubercular Pleurisy. 

This is characterized especially by an increase, both relative and absolute, 
in the number of lymphocytes. A pleural lymphocytosis exists when there 
is an excess of mononuclear cells, with abundant protoplasm, a large nucleus, 
and smaller than the endothelial cells. In the very early days of the infection 
a neutrophilia may exist, but this is rarely seen, as attention may not be 
drawn to the condition sufficiently early. Associated with these polynuclear 
cells in the early stage we may find an increase in the number of endothelial 
cells. Eosinophile cells are frequently observed, but do not have any definite 
relation to tuberculosis as an infection.^ The red cells may be occasionally 
numerous, but are usually small in number. ^ 
Secondary Tubercular Pleurisy. 

As a rule, tubercular pleurisy secondary to a pulmonary tuberculosis yields 
a liquid which is poor in cells, practically all of which are very much altered 
and in some cases very difficultly recognizable. The polynuclear types may 
predominate to such an extent that a distinct polynucleosis exists as evidence 
of a septic rather than a true tubercular pleurisy. The polynuclear cells are 
usually old, much deformed, and their nature recognizable only by staining 
their neutrophile granules. Where the infection is directly tubercular and 
not mixed (the latter, however, usually being the case), an approximately 
equal division of the polynuclear and mononuclear forms may obtain. In 
this type of pleurisy the eosinophile cells may be very numerous, in one case 
of Widal and Ravaut constituting 54 per cent, of the cellular elements. 
The endothelial cells may be, as in the primary tubercular pleurisy, suffi- 
ciently numerous to constitute a distinct endotheliosis. They are, however, 
single and very rarely grouped in masses. 
Pneumococcus Pleurisy. 

This is a truly septic type of pleurisy and is characterized by a distinct 
polynucleosis.^ In the early stages of this type of pleurisy, the endothelial 
cells may be very numerous, while in the later stages they may be much dimin- 
ished. In this, as in all types of pus accumulations, marked autolysis is 
present so that the cells may show extreme degeneration. As this condition 
progresses toward recovery, some of the polynuclears may be replaced by the 
mononuclear lymphocytes; while if suppuration becomes extensive the poly- 
nuclears increase and autolysis becomes extremely marked. In such exudates 
the pneumococcus may be demonstrated by staining methods. 
Streptococcus Pleurisy. 

This type is especially associated with a polynucleosis. These cells are 
frequently observed undergoing karyolysis, the cell body usually being mark- 

1 Morris, Jour. Lab. and Clin. Med., 1916, I, 540. 

2 See Bullock and Twichell (Am. Jour. Med. Sc, 1915, CXLIX, 848) for a study of 
exudates in artificial pneumothorax. 

3 Evans, Jour. Infect. Dis., 1916, XIX, 440; Weiss, Arch. Int. Med., 1919, XXIII, 395. 



PLATE XXXV. 




Exudate from Tubercular Pleurisy. (Eosin-hematoxylin Stain ) 



\i 



PLATE XXXVI. 




Exudate in Pneumonic Pleurisy. (Eosin-hematoxylin Stain.) 



TRANSUDATES AND EXUDATES 751 

edly degenerated. In its early stage it may be accompanied by an endothelio- 
sis, but when well developed is usually associated with the presence of only a 
few isolated endothelial cells. Stained smears show streptococci in large 
numbers. 

Typhoid Pleurisy. 

In this type a lymphocytosis is usually observed along with an endothe- 
liosis, these latter cells being in large masses instead of in single isolated forms 
as observed in the secondary tubercular pleurisy.^ This point may be valu- 
able in differentiating these two conditions, which are associated with an in- 
crease in the number of lymphocytes. The eosinophiles may be increased 
and red cells may be present in large numbers. The identification of the 
specific organism will serve as a positive differentiation from tubercular 
conditions. 

Malignant Pleurisy. 

This is a type of the aseptic pleurisies and may accompany malignant 
growths of the lung or pleura. Nothing characteristic is found in the cytolo- 
gic formula of such exudates, but occasionally portions of the tumor mass 
may be obtained or certain cells may be found which are more or less distinc- 
tive, although not absolutely pathognomonic. These cells are frequently 
confused with the larger endothelial cells of the pleura, but are characterized, 
according to Deguy and Guillaumin, as follows: Mahgnant pleural cells con- 
tain glycogen which is recognized by the brown coloration shown on treat- 
ment with dilute iodin solutions, they contain large amounts of fatty material, 
they are extensively vacuolated, these vacuoles may be large or so numerous 
and small that the cell resembles a sponge, the cells are extremely large, and 
the nucleus usually shows mitotic figures.^ 

Nephritic and Cardiac Pleurisy. 

The secondary exudate observed in cardiac and renal conditions is char- 
acterized especially by the marked endotheliosis. These endothelial cells are 
grouped in masses of 5 to lo cells, which show more or less degeneration, their 
contours and their large nuclei being distinct. This endotheHosis is not com- 
plicated by the presence of many other cells in the pure nephritic pleurisy, 
but in the cardiac type we usually find a polynucleosis at the same time. 
This polynucleosis is much more marked where infarcts or emboli have com- 
plicated the condition than when it is due to a pure congestion. Numerous 
red cells are especially observed in association with a congestive pleurisy. 

Peritoneal Exudates. 

The cytological examination of peritoneal exudates has, as yet, yielded 
fewer diagnostic points than has that of pleural exudates.^ It is, however, 
sometimes possible to differentiate a tubercular peritonitis from an ascites or 

1 See Pepper, Am. Jour. Med. Sc, 1916, CLI, 663. 

2 See Warren, Arch. Int. Med., 1911, VIII, 648; Josefson, Ztschr. f. klin. Med., 1916, 
LXXXII, 331; Judd, Am. Jour. Med. Sc, 1917, CLIII, 717. 

3 See Szecsi, Folia Haemat., 1912, XIII, i. See RoUeston (Brit. Med. Jour., 1914, I, 
238) and Weishaupt (Arch. f. Gynak., 1914, C, 496) for a discussion of eosinophilia in 
peritoneal exudates; also, Carslaw, Brit. Jour. Surg., 191 5, III, i. 



l!(l| 



752 DIAGNOSTIC METHODS 

an ovarian cyst by means of such examinations. Tubercular peritonitis 
usually shows a lymphocytosis and also a relative polynucleosis. A few 
endothelial cells may occasionally be found, but these do not yield much in- 
formation. In ascites of hepatic origin few cellular elements are observed 
beyond the peritoneal endothelial cells. In ovarian cysts there are fewer 
cellular elements as a rule, but those present are usually large, round, or oval, 
and filled with vacuoles. Moreover, cylindrical ciliated epithelial cells as 
well as goblet cells or red cells are frequently relatively numerous. 

f V. Cyst Fluids 

(i) Ovarian Cysts. 

(a) Serous Cysts. 

Such cysts are true retention cysts formed by dilatation of the Graafian 
follicles and retention of the ovarian secretion and are known as Hydrops 
foUiculorum Graafii. They contain a clear, watery, serous liquid, which has 
an amber color, a specific gravity ranging between 1,005 and 1,022, and a 
chemical composition practically identical with that of other serous fluids. 

(b) Myxoid or Colloid Cysts. 

These are proliferating cysts developed from the epithelial tubules. " We 
sometimes find in small cysts a semisolid, transparent, or somewhat cloudy 
or opalescent mass which appears like solidified glue or quivering jelly, and 
which has been called colloid because of its physical properties. In other 
cases the cysts contain a thick, tough mass which can be drawn out into long 
threads, and, as this mass in the different cysts is more or less diluted with serous 
liquids, their contents may have a variable consistency. In other cases the 
small cysts may also contain a thin watery fluid. The color of the contents 
is also variable. In certain cases it is bluish-white, opalescent, and in others 
yellow, yellowish-brown, or yellowish with a shade of green. They are often 
colored more or less chocolate-brown or reddish-brown, due to the decomposed 
blood pigment. The reaction is alkahne or nearly neutral. The specific 
gravity, which may vary considerably, is generally 1,015 to 1,030, but may be, 
in a few cases, 1,005 to 1,010 or 1,050 to 1,055. Though the contents of the 
proliferating cyst may have a variable composition, still it may be character- 
ized, in typical cases, by its slimy or ropy consistency; by its grayish-yellow, 
chocolate-brown, sometimes whitish-gray color; and by its relatively high 
specific gravity. Such a liquid does not ordinarily show a spontaneous 
coagulation" (Hammarsten) . 

Microscopical examination of the sediment shows red and white blood- 
cells, large epithelial cells, which may be filled with vacuoles, cylindrical or 
goblet cells, granular cells showing more or less fatty degeneration, fatty 
granules, cholesterin crystals, and colloid corpuscles in the form of large, 
circular, highly refractile bodies.^ 

Chemically, these cysts are characterized by the presence of colloid, which 

1 Dienst (Miinch. med. Wchnschr., 191 2, LIX, 2731) has called attention to the fact 
that fibrin is absent in the fluid from ovarian cysts while it is constantly present in ascitic 
fluids. 



PLATE XXXVII. 




Exudate in Malignant Pleurisy. (Eosin-hematoxylin Stain. After Deguy and 

guillaumin.) 



ill 
III 



TRANSUDATES AND EXUDATES 753 

is not a distinct chemical entity. It is a gelatinous substance, insoluble in 
water and acetic acid, soluble in alkalies, and yields a reducing body on boiling 
with acids. Not infrequently pseudomucin (metalbumin) is found, especially 
in the extremely viscid fluids. For its detection the serum albumin must be 
previously removed by the addition of acetic acid, boiling, and filtering. The 
filtrate is treated with alcohol when a thready precipitate forms. If this 
precipitate be boiled with HCl, a substance is formed which reduces copper 
solutions quite markedly. Pseudomucin is distinguished from true mucin by 
the fact that it is not precipitated by acetic acid. Mitjukoff has isolated a 
further colloid body from certain ovarian cysts, to which he gives the name of 
paramucin. It is precipitated by acetic acid and is soluble in an excess. If 
treated with alkali it first swells up and then dissolves in an excess of the re- 
agent. It differs from mucin and pseudomucin in the fact that it reduces 
copper solution without previous boiling with acids. McConnelP has 
recently reported the finding of a multi-locular cyst of the ovary, which 
contained true mucin. 

(c) Papillary Cysts. 

These are intrahgamentary types and contain a yellow, yellowish-green, 
or brownish-green fluid which contains only traces of pseudomucin. The 
specific gravity ranges between 1,032 and 1,036. 

{d) Dermoid Cysts. 

These may be seen in the shape of small cysts not larger than a pea, but 
usually they are much larger, in some cases reaching the size of a man's head. 
The cyst usually contains a fatty unctuous material, which is derived from 
the epidermal lining of the cyst, and associated with it fat, desquamated 
epithelial scales, hair, teeth, bone, cartilage, etc.^ 

(2) Parovarian Cysts. 

These cysts of the organ of Rosenmiiller contain a clear, pale yellow, or 
colorless, limpid fluid or occasionally one showing slight opalescence. The 
specific gravity ranges from 1,002 to 1,010 and differs from that of the ovarian 
cyst by its usual limitation to these lower figures. Albumin may be present 
in small amounts or be entirely absent, while pseudomucin is rarely, if ever, 
present. 

(3) Hydrocele. 

The contents of such a cyst are usually clear, show a color which may range 
from yellow to green, have a specific gravity of 1,015 to 1,030, and usually 
coagulate spontaneously. They contain a relatively large percentage of al- 
bumin, of which about 50 per cent, is globulin. In ordinary hydrocele many 
large oval cells may be seen, which have an eccentric nucleus and may be 
grouped in masses, although more frequently they appear as isolated ceUs; 
in some cases many eosinophiles, but this is rare in uncomplicated hydroceles. 
If the hydrocele be of tubercular origin a marked lymphocytosis usually exists. 

^ Jour. Med. Research, 1909, XX, 105. 

2 See Spalding, Am. Jour. Obs. and Dis. Women and Child., 1919, LXXX, 401. 
48 



754 DIAGNOSTIC METHODS 

(4) Spermatocele. 

Fluj[ds from such cysts are usually thin, colorless, and cloudy like thin 
milk. They may have an acid reaction, but are ordinarily alkaline. The 
specific gravity ranges between i ,006 and i ,01 o. Such fluids do not coagulate 
either spontaneously or after the addition of blood. Microscopically, one 
observes cell detritus, fat granules, leucocytes, and spermatozoa. 

(5) Hydronephrosis. 

This is a true retention cyst of the kidney, due primarily to obstruction of 
the ureter, which may be either congenital or acquired. Material aspirated 
from a renal cyst is usually clear, but may be yellowish or reddish and dis- 
tinctly turbid. Its specific gravity varies from 1,010 to 1,015, while the chem- 
ical composition is usually suggestive of urine. For some time the presence of 
urea and uric acid in hydronephrotic cysts was supposed to be pathognomonic, 
but it has been shown that these substances may be present both in ovarian 
and pancreatic cysts and may even be lacking in old renal cysts. Occasion- 
ally epithelial cells, derived from the urinif erous tubules may be found, but 
such cells are not always present nor are they sufficiently characteristic to 
be of great importance from the diagnostic standpoint. 

(6) Hydatid Cysts. 

The fluid obtained by puncture of an echinococcus cyst is usually clear and 
of an alkaline reaction, has a specific gravity varying between 1,005 ^^^ '^P^Sj 
is practically free from albumin, and contains a large amount of inorganic 
salts, especially of sodium chlorid. The characteristic findings of such a 
cyst are the booklets, scolices, and shreds of faintly laminated membrane. In 
some cases no trace of any morphological elements can be found, hut usually 
the diagnosis is rendered certain, especially if careful search be made, by the 
presence of some portion of the parasite or the cyst membrane. 

(7) Pancreatic Cysts. 

The puncture fluid from a pancreatic cyst varies in its physical properties, 
depending upon the nature of the cyst as well as the length of time the fluid 
has remained in the cyst. It is usually bloody in character, has a specific 
gravity ranging from 1,010 to 1,030 and may contain methemoglobin, hema- 
tin, and cholesterin. As characteristic constituents of such a cyst one finds 
ferments, which will digest all types of food material.^ Such tests may be 
performed as outlined in the sections on Gastric Contents and Feces. 

VI. Cerebrospinal Fluid 

Since the introduction of lumbar puncture by Quincke, the cerebrospinal 
fluid has gained more or less importance from the diagnostic point of view. 
So much may be learned, either from the standpoint of direct or differential 

1 See Christian (Arch. Int. Med., 191 2, IX, 143) who reports peculiar findings in the 
fluid of an epigastric cyst; also Besley, Jour. Am. Med. Assn., 1914, LXII, ion; Ipsen, 
Hospitalstid., 1914, LVII, 889 and 921; Speese, Ann. Surg., 1914, LX, 673; Rosenthal, 
Arch. f. Verdauungskr., 1914, XX, 619; Eustachio Rif. Med., 1915, XXXI, 424; Willis 
and Budd, Surg., Gyn. and Obs., 1915, XX, 688. 



TRANSUDATES AND EXUDATES 755 

diagnosis, that every practitioner should be able to perform a lumbar puncture 
and to examine the fluid obtained. There is little danger in the procedure 
as the spinal cord does not reach to the point of puncture and the fibers of the 
Cauda equina are sufficiently movable to escape the needle. While few bad 
effects are observed in the ordinary run of cases, a few have been reported in 
which symptoms of collapse were evident. It should be a rule, therefore, 
to stop proceedings if such symptoms arise and also to keep the patient quiet 
in bed for at least 24 hours following the puncture, so that the pressure in the 
cerebrospinal cavity may become equalized.^ 

Lumbar Puncture. 

The patient is placed upon his left side near the edge of the bed, the knees 
should be flexed upon the abdomen, and the site of puncture prepared as for 
any surgical procedure. The needle used for puncture should be from 5 to 
10 cm. long and have a lumen from i to 2 mm. in diameter. It is always wise 
to provide the larger needles with a stylet, so that tissue fragments or blood 
may not gain entrance accidentally to the tube and thus lead to possible 
diagnostic errors. This stylet may be removed the moment the needle 
penetrates the dura mater. 

The site of puncture should be on a level with the junction of the third 
and fourth lumbar vertebrae at a point about i cm. to the side (preferably 
the upper) of the median line. The needle is directed slightly upward and 
inward, the depth to which the puncture should be made varying with the 
age of the patient, the younger the child the less the depth. This puncture 
should be made carefully and yet with sufficient force to penetrate easily 
the musculature. Tf any marked resistance arises, it is probable that the 
needle has struck the vertebra, in which case the pressure must be reduced or 
the needle may break. This is not an infrequent occurrence with those not 
used to the technic, so that it may be advisable for the student to practice 
the procedure upon the cadaver. As soon as the dural sac is reached, the 
cerebrospinal fluid will flow from the canula, the rate of flow indicating in a 
general way the pressure of the fluid. No aspiration should be used at any 
time, as this procedure is extremely dangerous. 

It is frequently advisable to know exactly what this pressure is, so that 
one may resort to the following method as used by Sahli. As soon as the 
needle penetrates the dura, a connection is made with a mercury manometer 
by means of a rubber tube filled with a i per cent, solution of carbolic acid. 
The portion of the manometer above the level of the mercury, forming the con- 
nection between it and the carbolic acid tube, must also be filled with the 
fluid. The manometer is filled with mercury to the zero point and held in 
such a manner that this point is on a level with the point of the aspirating 

^ For discussions regarding the source of the spinal fluid and variations in it see Dixon 
and Halliburton, Jour. Physiol., 1913, XLVII, 215; Ibid., 1914, XLVIII, 129 and 317; 
Dandy and Blackfan, Jour, Am. Med. Assn., 1913, LXl, 2216; Frazier, Ibid., 1914, LXIII, 
287; Gushing, Jour. Med. Research, 1914, XXXI, i; Weed, Ibid., 21, 51 and 93; Wegefarth, 
Ibid., 119 and 149; Wegefarth and Weed, Ibid., 167; Frazier and Peet, Am. Jour. Physiol., 
1914, XXXV, 268; Weed and Gushing, Ibid., 1915, XXXVI, 77; Frazier, Jour. Am. Med. 
Assn., 1915, LXIV, 1119; Halliburton, Brain, 1916, XXXIX, 213; Lancet, 1916, II, 779; 
Bloch, Jour. A. M. A., 1917, LXVIII, 691. 



756 



DIAGNOSTIC METHODS 



needle, which is possible with ordinary manometers only when the connecting 
tube is of considerable length.^ Under normal conditions the dural pressure, 
in the dorsal position, ranges between 5 and 7.5 mm. of mercury, or 60 to 
100 mm. of water if a water manometer be used. In pathologic conditions, 
such as meningitis or brain tumor, it ranges between 15 and 60 mm. of 
mercury or 200 to 800 mm. of water. 

Normal cerebrospinal fluid is colorless, limpid, and free from morphologi- 
cal elements. Its specific gravity ranges between i ,002 and i ,010. 2 It is alka- 
line in reaction, the degree of alkalinity varying between 15 and 20.^ It con- 
tains a trace of protein and about o.i per cent, of glucose. The salt content of 

this fluid is, according to Zdarek, 0.836 
gram, of which 0.429 gram is referable 
to sodium oxid and 0.017 to potas- 
sium oxid. This would rather militate 
against the older statements that a 
large amount of potassium salts as 
compared with sodium salts was pres- 
ent. The relation of KCl to NaCl is, 
according to Nawratzki, i to 18, while 
Zdarek gives this ratio as i to 40. 
The former figure agrees closely with 
that obtained by various workers with 
pathological cerebrospinal fluids.^ 
The amount .of fluid obtained by 
lumbar puncture is extremely variable. 
In normal individuals this amount is 
unknown as puncture is rarely per- 
formed upon normal cases. Patholog- 
ically, the amount obtained varies be- 
tween 10 and 100 c.c. Naturally, if 
the communication between the subarachnoid spaces of the brain and of the 
spinal cord is blocked by a tumor or inflammatory adhesions, or if the aque- 
duct of Sylvius or the foramen of Magendie be obliterated, little fluid may 
be obtained by puncture, although large amounts may be present above the 
obstruction. The largest amounts are seen in cases of serous or tubercular 
meningitis, so that such conditions may usually be ruled out if a small amount 
of fluid is obtainable. 

Pathologically, we may observe a very cloudy fluid, due to the presence 




-Lumbar puncture: a, 
Quincke's site; b, Maran's site; c, Chipault's 
site. {Tyson.) 



^ See Strauss (Jour. Am. Med. Assn., 1914, LXII, 1327) for a special spinal-puncture 
needle with manometer attachment. 

2 See Stanford (Ztschr. f. physiol. Chem,, 1913, LXXXVI, 43 and 219), who shows that 
this density is higher in progressive paralysis and epilepsy than in other conditions. 

^Levinson (Jour. Infect. Dis., 1917, XXI, 556) shows that the H-ion concentration (Ph) 
of the spinal fluid in nonmeningitic cases varies between 7.4 and 7.6. See, also, Levinson, 
Arch. Pediat., 1916, XXXIII, 241; Hurwitz and Trouter, Arch. Int. Med., 1916, XVII, 
828; Felton, Hussey, and Bayne- Jones, Ibid., 191 7, XIX, 1085. 

^ See Rosenbloom and Andrews, Arch. Int. Med., 1914, XIV, 536; Halverson and Berg- 
eim (Jour. Biol. Chem., 191 7, XXIX, 337) discuss the calcium content of the cerebro- 
spinal fluid. 



TRANSUDATES AND EXUDATES 757 

of leucocytes, erythrocytes, and endothelial cells. This cellular admixture 
may be so extensive that the fluid resembles pure pus. In cases of cerebral 
hemorrhage from the ventricles, hemorrhagic pachymeningitis, or traumatic 
lesions of the spinal cord, so much blood may be present as to give the appear- 
ance of practically pure blood, the color varying from a bright red to a brown- 
ish or greenish-red, depending upon the length of time it has remained in con- 
tact with the remaining portion of the fluid. This admixture with blood 
may lead to the spontaneous coagulation of the fluid. ^ This may serve as a 
differentiating point between inflammatory and noninflammatory lesions. 
Thus, in tubercular meningitis very slight coagulation may be observed while 
in the epidemic cerebrospinal meningitis the coagulum may be very firm. 

The chemical examination of the cerebrospinal fluid offers some points of 
clinical value. ^ While the albumin content normally is much less than o. i per 
cent., it may vary under pathologic conditions to as high as 0.8 per cent. The 
total protein, especially the euglobulin portion, is increased in all cases of 
acute exudative inflammations of the meninges, in hydrocephalus as Polanyi^ 
has shown and, also, in syphilitic and parasyphilitic diseases of the cere- 
brospinal tract. Glucose is usually present but may entirely disappear under 
pathologic influences due to the autolysis controlled by the leucocytic fer- 
ments, the glucose being converted into lactic acid.^ Cholin is present nor- 
mally in traces, while pathologically it may vary, according to Donath, 
between 0.021 per cent, and 0.046 per cent. 

Koch^ has introduced the following method for examination of this fluid, 
which should extend our knowledge. The fluid is centrifuged and the sedi- 
ment used for the bacteriological and cytological examinations. Ten c.c. of 
this clear fluid are placed in a beaker, 3 c.c. of a saturated solution of ammo- 
nium nitrate and 5 drops of nitric acid are added, and the mixture heated on a 

^ Schwarz (Deutsch. Ztschr. f. Chir., 1913, CXXIV, 346) shows that a yellowish tint 
(xanthochromasia) is frequently due to conversion of the blood pigment into urobilin and, 
hence, that this is a valuable indication of intracranial hemorrhage. See, also, Babes, 
Compt. rend. Soc. de biol., 1914, LXXVII, 67; Hanes, Am. Jour. Med. Sc, 1916, CLII, 
66; Sprunt and Walker, Bull. Johns Hopk. Hosp., 1917, XXVIII, 80. For total nitrogen 
and urea of spinal fluid see Adler and Ragle, Boston, Med. and Surg. Jour., 1914, 
CLXXI, 769; Woods, Arch. Int. Med., 1915, XVI, 577; and Cullen and Ellis, Jour. 
Biol. Chem., 1915, XX, 511; Kahn, Ibid., 1916, XXVIII, 203. Weston (Jour. Med. 
Research, 1915, XXXIII, 119), discusses the cholesterol variations in this fluid. 

2 See Cantieri, Rif. Med., 191 6, XXXII, 977 and 1009; Kahn and Neal, Proc. Soc. 
Exper. Biol, and Med., 1916, XIV, 26; Johnston, Am. Jour. Dis. Child., 1916, XII, 112; 
Leopold and Bernhard, Ibid., 1917, XIII, 34; Levinson, Am. Jour. Dis. Child., 1919, XVIII, 
568. 

3 Biochem. Ztschr., 1911, XXXIV, 205. See, also, Ferrier, Lancet, 1913, II, 1107; 
Ayer and Viets, Jour. A. M. A., 1916, LXVII, 1707; McGregor, Jour. Biol. Chem., 191 7, 
XXVIII, 403; Novick, Jour., Infect. Dis., 1917, XXI, 52;Lecene, Mestrezat and Boutilier, 
C. R. soc, biol. Paris, 1918, LXXXI, 597. 

"* See Kopetzky (Trans. Am. Acad, of Ophth. and Oto-Laryngol., 1912, I, 261) who 
believes the absence of the copper-reducing body to be the earliest sign of bacterial invasion 
of the central nervous system. See Szabo, Ztschr. f. d. ges. Neurol, u. Psychiat., 1913, 
XVII, 145; Soper and Granat, Arch. Int. Med., 1914, XIII, 131; Hopkins, Am. Jour. Med. 
Sc, 1915, CL,847; Schloss and Schroeder, Am. Jour. Dis. Child., 1916, XI, i; Rieger and 
Solomon, Boston Med. and Surg. Jour., 1916, CLXXV, 817; Kraus and Corneille, Jour. 
Lab. and Clin. Med., 1916, I, 685; Kiely, Ibid., 191 7, II, 645; Weil, C. R. Soc biol. Paris, 
1918, LXXXI, 364; Mestrezat, Weissenbach and Boutlier, Ibid., 655; Weissenbach, Bull, 
et Mem. Soc. Med. hop. de Paris, 1918, XXXIV, 113. 

^Arch. Neurol., 1907, III, 331; Arch. Neurol, and Psychiat., 1909, IV, i; Koch and 
Koch, Jour. Biol. Chem., 191 7, XXXI, 395. 



758 



DIAGNOSTIC METHODS 



water-bath until complete coagulation has occurred. This precipitate of 
protein^ is filtered through a perforated Gooch crucible, washed with o.i per 
cent, ammonium nitrate solution and then with alcohol, dried at ioo°C. 
and weighed. The filtrate is taken before the washing with alcohol is begun, 
concentrated to lo to 15 c.c. and 2 c.c. of i per cent, barium nitrate solution 
added while boiling. The barium sulphate precipitate is allowed to settle 
over night, is filtered off, dried and weighed. With the filtrate a phosphorus 
estimation is made by the molybdic method, with subsequent precipitation 
as magnesium ammonium phosphate in the usual way. Increase in the 
phosphorus and sulphur content is associated with degenerative changes in 
the nerve tissue. A test for reducing substances is made with i -c.c. of the 
original fluid. / 

Microscopic Examination. 

By far the most important part of the clinical examination of cerebro- 
spinal fluid is a study of the bacteriology and cytology of the fluid. Normally 
the fluid contains practically no morphologic elements, while under patho- 
logic conditions large numbers of various types of cells may be present. The 
material for examination is obtained as previously described in the section 
on cytology. In true tubercular meningitis a lymphocytosis is almost inva- 
riably observed, while in the epidemic type, due to the meningococcus of 
Weichselbaum, the cells are of the polynuclear type. In the chronic cases of 
epidemic meningitis as well as during convalescence from this disease, the 
lymphocytes may be present to such an extent that a slight degree of lympho- 
cytosis is present, but never to the same extent as observed in the tubercular 
type. 2 A lymphocytosis is also observed in syphilitic lesions of the central 
nervous system. This is important from the standpoint of differential diag- 
nosis. In certain cases of brain tumor, of non-specific origin, a lymphocyto- 
sis may obtain along with an increase in the protein-content of the fluid. 
In the meningitis due to the pneumococcus, a polynucleosis is the rule, al- 
though occasional cases are seen in which a lymphocytosis obtains.^ In the 
prodromal stages and early days of acute anterior poliomyelitis a marked 
increase of fluid is accompanied by an increase in number of cells, which are 
largely of the mononuclear type. These are replaced by the polymorphonu- 
clear forms as the number diminishes. In the microscopic examination 
of the cerebrospinal fluid it is sometimes of interest to know the number of 
cells per c. mm., of the fluid. The technic is as follows: The following 
mixture is employed for staining the white and dissolving the red cells: 
Methyl violet 0.2 gram, glacial acetic acid 5 c.c, water to 100. Fill the 
pipet with stain to the mark i and then fill to point 11 with uncentrifuged 
cerebrospinal fluid. Shake thoroughly, let stand for five minutes and count, 

^ Mott advises the following method for protein: 10 c.c. of the centrifuged fluid are 
faintly acidified with acetic acid and 20 c.c. of absolute alcohol are added. The mixture is 
boiled and filtered, the precipitate of protein being dried and weighed. 

2 Wassermann andLange (Berl. kUn. Wchnschr., 1914, LI, 527) believe that these lym- 
phocytes are, at least, one source of the substances responsible for the positive Wassermann 
reactions obtained with spinal fluid. 

^ See, however, Brady, Jour. Am. Med. Assn., 1913, LX, 972. 



TRANSUDATES AND EXUDATES 759 

using a Fuchs-Rosenthal counting chamber. The ruled surface of the cell 
contains 3.2 c. mm. of fluid. Count all of the white cells in the entire ruled 
area, multiply by 11 and divide by 32. The result is the number of cells per 
c. mm., which varies from i to 10 normally.^ 

Epidemic Cerebrospinal Meningitis. 

In these cases the fluid may be transparent, but is usually somewhat 
opalescent and may be thick and purulent. ^ The cellular elements are usually 
polynuclear in type and red cells may be more or less numerous. 

Smears made from the sediment show the presence of numerous diplococci, 
which closely resemble the gonococcus in morphological and staining char- 
acteristics. This organism, the diplococcus meningitidis intracellularis of 
Weichselbaum, appears as a diplococcus, _ ^ 

each element forming a hemisphere with " ^^ •' 

its parallel side contiguous to that of its ^^^ «^ 

mate.^ It is sometimes seen in the form ^ ' «" ,/ ^^ " ^ 
of tetrads or as isolated cocci, which 
appear as true spheres of variable size and 
showing a clear space in their interior. 
It is stained with the ordinary dyes and is 
negative to Gram's stain. For its cultural 
peculiarities see the last chapter of this 
book. Not infrequently one observes 

specimens of the meningococcus which . ^ 

, „ . . . , . Fig. 159. — Diplococcus intracellularis 

show a Gram-positive reaction, so that it meningitidis. (Councilman.) 

is difficult to distinguish them, especially 

when they are in the form of isolated cocci or in groups of two or four, from 
the pneumococcus. This type has been described as the meningococcus of 
Bonome, while Jaeger and Heubner describe a diplococcus which is Gram- 
positive and may be confused with the unusual types of Weichselbaum's 
meningococcus. 

Recently Flexner has succeeded in preparing an antimeningococcic serum 
which appears to have remarkable results in controlling this hitherto un- 
manageable disease. In using this serum, the injections must be made 
directly into the spinal cavity.* More or less frequent injections of the 

^ See Blatteis and Lederer, Jour. Am. Med. Assn., 1913; LX, 811; Karpas, Ibid., 1913, 
LXI, 262; Engman, Buhman, Gorham and Davis, Ibid., 735; Brem, Ibid., 742; Maruyama, 
Wien. klin. Wchnschr., 1913, XXVI, 1233; Ball, Interstate, Med. Jour., 1913, XX, 1109; 
Rubenstone, New York Med. Jour., 1913, XCVIII, 1210; Gordon, Ibid., 1914, XCIX, no; 
Mitchell, Darling and Newcomb, Jour. Nervous and Ment. Dis.; 1914, XLI, 686; Cottin, 
Rev. med. de la Suisse Rom., 1914, XXXIV, 715; Bigelow, Cleveland Med. Jour., 1915, 
196; Pollock, Trans. Chic. Path. Soc, 1916, X, 43; Larkin and Cornwall, Jour. Lab. and 
Clin. Med., 1919, IV, 352; Herrick and Dannenberg, Jour. A. M. A., 1919, LXXIII, 1321, 

2 Nobecourt and Peyre, Presse Med., 1916, XXIV, 461, show that the protein content 
of the spinal fluid is persistently high as long as the infection persists. See Tashiro and 
Levinson, Jour. Infect. Dis., 1917, XXI, 571. 

2 See Frost, Pub. Health Rep., 1912, XXVII, 97;Broers and Smit, Nederl. Tijdschr. v. 
Geneesk., 1915, II, 1175; also, Hort, Lakin and Benians (Brit. Med. Jour., 1915, i, 541), 
Foster (Ibid., 543) and Donaldson (Lancet, 1915, i, 1333), who suggest that the meningococ- 
cus is only a phase in the hfe history of the true infective agent. 

^ See Dubois, Jour. Am. Med. Assn., 1913, LX, 820; also, WoUstein, Jour. Exper. Med., 
1914, XX, 201; Dubois and Neal, Am. Jour. Dis. Child., 191 5, IX, i. 




760 DIAGNOSTIC METHODS 

serum and examinations of the lumbar fluid are made and the influence of 
the serum estimated by bacteriological and cytological examinations. In 
the cases of mixed cerebral infection, in which the meningococcus is asso- 
ciated with the pneumococcus, streptococcus, typhoid bacillus, staphylo- 
coccus, and other organisms, this serum does not seem to have as much 
influence as in the pure meningococcus infections.^ 

It is to be remembered that a purulent meningitis may be secondary 
to infection with practically all of the pus-forming organisms found within 
the system. It is, therefore, essential that any infection showing meningeal 
symptoms should be investigated by an examination of the cerebrospinal fluid. 

In sleeping sickness, a study of the cerebrospinal fluid frequently reveals 
the presence of the trypanosoma Gambiense. These parasites are not found 
in all cases, but when present usually furnish a grave prognosis. 

Recent investigation of the cerebrospinal fluid, applying Wassermann's 
serum reaction for syphilis, has shown that in the large majority of nervous 
cases of syphilitic origin a positive reaction is obtainable. 

Tubercular Meningitis. 

The fluid in such cases is usually clear, but may be slightly opalescent. 
The cellular elements are largely mononuclear while a few red cells may be 
present. If tubercle bacilli cannot be found after careful search, the presence 
of a lymphocytosis along with an increased protein-content will be at least 
suggestive, while animal inoculation or the tuberculin test will clear up the 
diagnosis in most cases. (See Lucas. 2) 

Acute Anterior Poliomyelitis. 

This disease has been definitely established as infectious through the work 
of Flexner and his associates. The causative agent was known to belong to 
the ultra-microscopic type and to pass the Berkefeld filter. In other words it 
is a filterable virus, the point of ingress and egress of which is the nasal and 
pharyngeal mucosa. This virus is transmitted by means of the biting stable- 
fly (stomoxys calcitrans) and probably, also, by the common fly and bedbug.^ 

Cultivation of this organism has been accomplished by Flexner and 

Noguchi.^ The culture medium consists essentially of sterile human ascitic 

fluid to which has been added a fragment of sterile fresh kidney tissue of the 

normal rabbit. Solid media is made by the addition of 2 per cent, agar to 

above. The exclusion of oxygen is necessary for the initial culture, but it 

^ Collignon and Pilod (Presse med., 191 1, XIX, 732) advocate the following test to 
differentiate meningococcic from other types of meningitis. To a few c.c. (50 drops) 
of centrifuged spinal fluid add one to four drops of antimeningococcus serum and incubate 
the mixture (along with a control tube containing only spinal fluid) at 56°C. for 12 hours. 
If the serum and spinal fluid be homologous, a definite precipitation ^^ill be observed. 
This test appears to be specific. 

2 Am. Jour. Dis. Child., 191 1, I, 230; also, Mandelbaum, Deutsch. Arch. f. klin. Med., 
1 913, CXIII, 92; Roby, Jour. x\m. Med. Assn., 191 5, LXV, 1027; Kasahara, Am. Jour. 
Dis. Child., 191 7, XIII, 141. 

3 Rosenau, Discussion in 15th Cong, on Hyg. and Demog., Abs. in Jour. Am. Med. Assn., 
i9i2,LIX, 1314; Anderson and Frost, Pub. Health Rep., 1912, XXVII, 1733; Howard and 
Clark, Jour. Exper. Med., 191 2, XVI, 850; Mitzmain, Pub. Health Rep., 1913, XXVIII, 
345; Lucas and Osgood, Jour. Am. Med. Assn., 1913, LX, 1611; Rosenau, Ibid., 1612; 
Sawyer and Herms, Ibid., 1913, LXI, 461. 

^ Jour. Exper. Med., 1913, XVIII, 461. See, also, Amoss, Ibid., 1914, XIX, 212. 



TRANSUDATES AND EXUDATES 76 1 

suffices to cover the media with a deep layer of sterile paraffin oil. Films 
prepared from the lower layers of the fluid cultures are made, are air-dried 
and fixed in methyl alcohol for one hour. Wash in distilled water and im- 
merse in Giemsa's solution (i drop of stain to i c.c. of distilled water) for two 
to twelve hours. Examination reveals minute globoid bodies arranged in 
pairs or short chains, or in small aggregated masses. These bodies are 
stained violet and average about 0.2 micron in diameter. Their pathogen- 
icity has been proven by inoculation of monkeys. 

The changes in the spinal fluid, while not absolutely diagnostic are sugges- 
tive. The amount of fluid usually increases early in the disease, this increase 
being associated with a relative increase in the mononuclear types. These cells 
may first be of the small lymphocytic variety but they soon changed to the large 
mononuclear^ orm. As the disease progresses, the cells diminish in number, the 
polymorphonuclear forms assuming the ascendency. The total protein is in- 
creased in amount (as detected by the Noguchi or Ross- Jones method), while 
fibrin formation appears to be an early sign, although this may soon disappear.^ 

Cerebrospinal Syphilis. 

Although such cases do not show pathognomonic findings in the cerebro- 
spinal fluid, yet certain factors are of importance in diagnosis. A lymphocy- 
tosis is usually observed together with a marked increase in the globulin con- 
tent, which is estimated as follows : ^ 

^ See "A Clinical Study of Acute Poliomyelitis" by Peabody, Draper and Dochez, 
published by the Rockefeller Institute in 1912; Flexner, Science, 1912, XXXVI, 685; 
Fraser, Jour. Exper. Med., 1913, XVIII, 242; Kling andLevaditi, Ann. de I'lnst. Pasteur, 

1913, XXVII, 718; Cassel, Deutsch. med. Wchnschr., 1913, XXXIX, 2507; Lust and Rosen- 
berg, Miinch. med. Wchnschr., 1914, LXI, 121; Flexner and Amoss, Jour. Exper. Med., 

1914, XIX, 411; Ibid., 1914, XX, 249; Ibid., 1915, XXI, 509; Flexner, Noguchi and Amoss, 
Ibid., 91; Flexner, Bull. Johns Hopkins Hosp., 1915, XXVI, 180; Am. Jour, Dis. Child., 

1915, IX, 353. See, also, Morse, Boston Med. and Surg. Jour., 1914, CLXX, 373; Corbus, 
Jour. Am. Med. Assn., 1914, LXIII, 550; Neisser. Berl. klin. Wchnschr., 1915, LII, 486; 
Abramson, Am. Jour. Dis. Child., 1915, X, 344; Neal and Dubois, Am. Jour. Med. Sc, 1916, 
CLII, 313: Flexner, Ibid., 1917, CLIII, 157; Kolmer, Arch. Pediat, 1917, XXXIV, 413; 
Neal, N. Y. Med. Jour., 1916, CIV, 167; Klein, Ibid., 219; Flexner, Jour. A. M. A., 1916, 
LXVII, 279; Hoyne and Cepelka, Ibid., 666; Rosenow, Towne, and Wheeler, Ibid., 1202; 
Nuzum and Herzog, Ibid., 1205; Nuzum, Ibid., 1437; Mathers and TunniclifE, Ibid., 1915; 
Nuzum, Ibid., i9i7,LXVIII, 24; Abramson, Ibid., 546; Gauss, Ibid., 779; Greeley, Boston 
Med. and Surg. Jour., 1917, CLXXVI, 540; Jour. Lab. and Clin. Med., 1917, III, 671; 
Kolmer, Freese, Matsunami and Meine, Am. Jour. Med. Sc, 191 7, CLIV, 720; Rosenow 
and Towne, Jour. Med. Res., 191 7, XXXVI, 175; Flexner and Amoss, Jour. Exper. Med., 
1917, XXV, 499, 525 and 539; Amoss and Taylor, Ibid., 507; Amoss, Ibid., 545; Bull, Ibid., 
557; Amoss and Chesney, Ibid., 581; Kolmer, Brown and Freese, Ibid., 789; Mathers, 
Trans. Chic. Path. Soc, 191 7, X, 145; Jour. Infect. Dis., 191 7, XX, 113; Hektoen, Mathers, 
and Jackson, Ibid., 1918, XXII, 89; Heist, Solis-Cohen, and Kolmer, Ibid., 169; Solis-Cohen 
and Heist, Ibid., 175 and 182; Rosenow and Wheeler, Ibid., 281; Solis-Cohen and Heist, 
Ibid., 175 and 182; Rosenow and Wheeler, Ibid., 281; Rosenow and Gray, Ibid., 345; 
Rosenow, Towne, and von Hess, Ibid., 313; McCann, Jour. Exper. Med., 1918, XXVII, 
31; Smillie, Ibid., 319; Tsen, Ibid., 1918, XXVIII, 269; Schultz and Dunnenberg, Arch. Int. 
Med., 1919, XXIII, 309; Flexner and Amoss, Jour. Exper. Med., 1919, XXIX, 379. 

2 See Kaplan and Casamajor, Arch. Int. Med., 191 2, IX, 262; also. Ball, Jour. Am. Med. 
Assn., 1912, LIX, 1272. Ellis (Jour. Exper. Med., 1913, XVIII, 162) asserts that no syphi- 
litic patient should be regarded as cured without a complete negative examination of the 
cerebrospinal fluid. Miller, Southern Med. Jour., 1915, VIII, 940; Collins, Am. Jour. Med. 
Sc, 1916, CLI, 222j Reasoner, Jour. A. M. A., i9i6,LXVI, 1917; Levin, N. Y. Med. Jour., 

1916, CIV, 212; With, Hospitalstid., 1916, LVIII, 1251, 1275 and 1311; Wile, Am. Jour. 
Syph., 191 7, I, 84; Babonneix and Javiller, Bull, et mem. soc. de Hop. de Paris, 1918, 
XXXIV, 15; Mclver, Jour. A. M. A., 1919, LXXIII, 1765; Larkin and Cornwall, Am. Jour. 
Syph., 1919, III, 76. 



762 DIAGNOSTIC METHODS 

Noguchi's Butyric Acid Test. 

One part (o.i or o.i c.c.) of spinal fluid is mixed with 5 parts (0.5 c.c.) of 
a 10 per cent, butyric acid solution in physiologic salt solution. This mixture 
is heated to boiling and immediately i part (o.i c.c.) of normal (4 per cent.) 
sodium hydrate solution is added and the mixture again boiled for a few 
seconds. The presence of an increased content of protein in the fluid is in- 
dicated by the appearance of a granular or flocculent precipitate which gradu- 
ally settles under a clear supernatant liquid. This precipitate appears with- 
in a few minutes in a specimen containing a considerable increase in protein, 
while two hours may be required to obtain a distinct reaction in specimens 
weaker in protein. May^ has called attention to the advisability of using 
larger portions of the fluid, preserving the same proportions of reagents as 
used by Noguchi. 

This reaction appears regularly in the cerebrospinal fluid of patients with 
syphilitic and parasyphilitic affections and also in all cases of inflammation of 
the meninges caused by such organisms as the meningococcus, pneumococcus, 
influenza bacillus, tubercle bacillus, etc. These latter cases are, however, 
easily differentiated from the syphilitic affections. Normal fluid gives a tur- 
bidity but the granular precipitate does not occur at all or only after many 
hours. 

Although this reaction is given in many non-syphilitic conditions, it has a 
certain value. It is not specific and, when present, does not necessarily indi- 
cate a syphilitic infection. On the other hand, it can be employed to estab- 
lish or confirm a deduction based upon the clinical history and the results of the 
Wassermann reaction and cytodiagnosis, thus becoming of great indirect 
diagnostic value. A syphilitic infection is, however, practically excluded by 
a negative reaction. In this respect this test has advantages over the Wasser- 
mann reaction, in w^hich a negative result is not always reliable as indicating 
absence of syphilitic infection. ^ 

Nonne's Test. 

This test,^ like the above, is for the detection of an increase of the protein 
material in the spinal fluid. It is, however, not limited to syphilitic condi- 
tions, although it is widely used for such cases. Nonne has advanced two 
steps which he styles "phases." 

Phase I. 

To a small portion of the clear cerebrospinal fluid add an equal volume of 
a hot saturated solution of ammonium sulphate. A trace of opalescence or a 
distinct turbidity, usually appearing within three minutes, indicates a posi- 
tive reaction. Normal fluids never show any appreciable change with this 
test, while pathologic types always react promptly. The test is positive in all 
infections of the nervous system and is of special interest in general paralysis, 

1 Arch. Int. Med., 191 1, VIII, 183. 

2 Nichols and Hough (Jour. Am. Med. Assn., 1913, LX, 108) report the demonstration 
of spirochaeta pallida in the cerebrospinal fluid and the successful inoculation of a rabbit 
therefrom. 

3 Arch. f. Psychiat. u. Nervenkrankh., 1907, XLIII, 433; Miinch. med. Wchnschr., 1907, 
LIV, 21 1 7. 



TRANSUDATES AND EXUDATES 763 

tabes and obscure cerebrospinal syphilis. Although not absolutely differ- 
ential, it is, yet, of great importance, as a negative test excludes syphilis 
and parasyphilis. 

Phase II. 

In case no turbidity is observed in Phase I, add a drop or two of acetic acid 
to the above mixture. A distinct turbidity appears at once. This phase is 
given, not infrequently, with normal fluids so that little value is attached to it. 

Ross- Jones Test. 

This^ is a modification of Phase I of Nonne and is, in my opinion, some^ 
what more delicate. Two c.c. of a saturated solution of ammonium sulphate 
are placed in a test-tube and i c.c. of the cerebrospinal fluid is gently run on to 
the surface so as to form a contact layer. A clear-cut, thin, grayish- white ring 
is observed at the junction of the liquids if the reaction be positive. This 
ring should appear within three minutes. The value of the test is the same 
as that above. 

Lange's Colloidal-gold Test. 

As neither the Nonne nor Ross-Jones tests exclude inflammatory processes 
in the brain or cord, a more differential test is to be desired. This seems to 
have been found in Lange's test. It is based on the fact that protein material 
precipitates colloidal gold at a dilution which is constant for definite proteins. 
If the protein be in less concentration a protective power over the colloidal 
particles is exerted and no precipitation occurs. In the case of spinal fluid, 
the protein present in different pathologic conditions shows a different precipi- 
tating point in syphilitic and non-syphilitic disorders. This test is, therefore^ 
much more differential than are the preceding ones given. 

Preparation of the Colloid Solution. 

Place 500 c.c. of double-distilled water in a Jena flask of i,ooo c.c. ca- 
pacity and heat slowly. When the temperature is about 6o°C., add, while 
still heating, 5 c.c. of i per cent, solution of gold chlorid (Merck's C. P.) and 
follow immediately with 5 c.c. of 2 per cent, solution of potassium carbonate. 
Heat rapidly to boiling and discontinue heating. Add quickly 5 c.c. of i per 
cent, dilution of formalin (not of formaldehyd). All these solutions must be 
added with a pipet as the amounts must be accurate in order to insure success 
in the preparation of the fluid. Shake the flask vigorously until a change of 
color occurs, beginning with a darkening of the fluid and passing through 
faint blue, dark blue, and purple to a final red. This is the finished colloidal 
solution and should be a distinct magenta and absolutely clear. Preserve in 
a dark-colored bottle. 

Technic. 

Place in a rack a series of twelve test-tubes. In the first, place with a 
pipet 1.8 c.c. of 0.4 per cent, freshly made sodium chlorid solution. In the 
other eleven tubes place i c.c. of the chlorid solution. To the first tube add 
0.2 c.c. of spinal fluid, which must be free from blood. The dilution in tube i 

^ Brit. Med. Jour., 1909, I, iiii. 






764 DIAGNOSTIC METHODS 

is, therefore, i to 10. Mix this thoroughly and place i c.c. of this first dilution 
in the second tube. This dilution is i to 20. Proceed in the same way with 
the remaining tubes, getting dilutions up to i to 20,440. 

After the dilutions are prepared, add to each tube 5 c.c. of the colloidal 
solution and let the tubes stand for 24 hours at room temperature. The read- 
ing of the test is as follows: The original tube showed a medium magenta 
shade. As the precipitation of the gold occurs, the color of the tubes con- 
stantly manifests a bluer tinge and finally becomes clear and colorless. The 
dilution at which the greatest precipitation occurs is noted and the results at 
various dilutions expressed by the + or — sign. Thus — indicates no 
precipitation; +, a red with beginning blue tinge; + +, red-blue and blue- 
red shades; + + +, violet and dark blue colorations; + + + +, light blue 
color ; + + + + +, complete precipitation with a clear and colorless solution. ^ 

In syphilitic and parasyphilitic cases the tendency of the precipitation is 
constantly toward the left of the series, that is toward the i to 10 dilution. 
The maximum effect in such conditions is i to 40 or i to 80. It never con- 
tinues past I to 640 and infrequently past i to 160. In non-syphilitic cases 
the tendency is toward the right of the series, that is toward the i to 20,000 
dilution, the maximum being observed at some point above i to 80. Tubercu- 
lous meningitis shows the most intense reaction at i to 160 or i to 320, while 
suppurative meningitis gives most marked reactions at i to 1,200 and up. 
This reaction promises to be of great value, especially in congenital syphilis. ^ 

^ Berl. klin. Wchnschr., 191 2, XLIX, 897; Ztschr. f. Chemotherap., 191 2, I, 44. See, 
also Grulee and Moody, Jour. Am. Med. Assn., 1913, LXI, 13; Eicke, Miinch. med. Wchn- 
schr., 1913, LX, 2713; Kaplan and McClelland, Jour. Am. Med. Assn., 1914, LXII, 511; 
Miller and Levy, Bull. Johns Hopkins Hosp., 1914, XXV, 133; Kafka and Rautenberg, 
Ztschr. f. d. ges. Neurol, u. Psychiat., 1914, XXII, 353; Lee and Hinton, Am. Jour. Med. 
Sc, 1914, CXLVIII, S3; Debenedetti, Rif. Med., 1914, XXX, 906; Brock, 111. Med. Jour., 

1914, XXVI, 422; de Crinis and Frank, Miinch. med. Wchnschr., 1914, LXI, 1216; Kafka, 
Ibid., 1915, LXII, 105; Solomon and Koefod, Boston Med. and Surg, jour., 1914, CLXXI, 
886; Solomon and Welles, Ibid., 1915, CLXXII, 398 and 625; Weston, Darling and New- 
comb, Am. Jour. Insan., 1915, LXXI, 835; Grulee and Moody, Am. Jour. Dis. Child., 1915, 
IX, 17; Barnes and Ives, Interstate Med. Jour., 1915, XXII, 792; Swalm and Mann, New 
York Med. Jour., 1915, CI, 719; Rubenstone and Schwartz, Ibid., 1273; Busquet, Bull. 
I'Acad. Med., 1915, LXXIV, 183; Miller, Brush, Hammers, and Felton, Bull. Johns Hopk. 
Hosp., 1915, XXVI, 391; Solomon, Koefed, and Welles, Boston Med. and Surg. Jour., 1915, 
CLXXIII, 956; Solomon and Welles, Ibid., 1916, CLXXIV, 50; Ramsay and Fidlar, Can. 
Med. Assoc. Jour., 1916, VI, 685; Oetiker, Ztschr. f. klin. Med., 1916, LXXXII, 235; 
Wilhams and Burdick, Colo. Med., 1916, XIII, 122; Weston, Jour. Med. Res., 1916, 
XXXIV, 107; Ibid., 191 7, XXXV, 367; Trimble, Jour. Lab. and Clin. Med., 191 7, IL 
199; Jeans and Johnston, Am. Jour. Dis. Child., 191 7, XIII, 239; Felton and Maxcy, Jour. 
A. M. A., 1917, LXVIII, 752; Hammes, Am. Jour. Med. Sc, 1917, CLIV, 625; Vogel, 
Arch. Int. Med., 1918, XXII, 496. 

2 Other methods for the detection of increased amounts of organic matter and protein 
in the cerebrospinal fluid have been introduced, but none of these have any advantage 
over those mentioned above Among these methods we find the Permanganate Reduction 
Index of Mayerhofer (Wien. klin. Wchnschr., 1910, XXIII, 651); in this connection see 
Hoffman and Schwartz, Arch. Int. Med., 1916, XVII, 293; the Permanganate Test of Bov- 
eri (Miinch. med. Wchnschr., 1914, LXI, 1215; see, also, Rubenstone, N. Y. Med. Jour. 

1915, CII, 1052; Lowrey, Boston Med. and Surg. Jour., 1917, CLXXVII, 115; Genoese, 
Policlinico, 1916, XXVI, 97; the Mastic Test of Emanuel (Berl. klin. Wchnschr., 1915, 
LII, 792; see, also, Langdon, Jour. Lab. and Clin. Med., 1918, III, 376; the precipitation of 
protein with sulphosalicylic acid as advocated by Pfeiffer, Med. Record, 1916, LXXXIX, 
66; and elaborated by Kirchberg, Deutsch. med. Wchnschr., 1918, XLIV, 657; and, recently 
the Lead Peroxid Reaction of Steinfeld, Jour. Lab. and Clin. Med., 1919, IV, 445; and the 
precipitation of the protein with an acidified solution of potassium dihydrogen phosphate, 
as advocated by Amoss, Jour. A. M. A., 1919, LXXII, 1289. 



TRANSUDATES AND EXUDATES 765 

BIBLIOGRAPHY 

1. Ad AMI. Principles of Pathology. Philadelphia, 1908. 

2. Kraus und Levaditi. Handbuch der Immunitatsforschung. Jena, 1908. 

3. Jordan. General Bacteriology. Philadelphia, 1908. 

4. Levinson. Cerebrospinal Fluid in Health and Disease, St. Louis, 1919. 

5. Mestrezat. Le Liquide Cephalo-Rachidien. Paris, 19 12. 

6. Oppenheimer. Handbuch der Biochemie. Jena, 1908. 

7. Plant, Rehm, and Schottmuller, Leitfaden zur Untersuchung der Zerebro- 

spinalflussigkeit. Jena, 1913. 



CHAPTER X 
SECRETION OF THE MAMMARY GLANDS 

I. General Considerations 

The normal secretion of milk takes place in the mammary glands of the 
female after delivery. It is true that a small quantity of milk may be secreted 
by the new-born of both sexes for a few days after birth. Moreover, cases 
have been reported in which the adult male secreted sufficient milk to act as 
a wet-nurse, but these must be regarded as cases of extreme rarity. 

During the course of a normal pregnancy a small amount of a thin, yellow- 
ish fluid may be expressed from the mammary glands, but as a rule the first 
real secretion is observed following delivery of the child. This secretion is 
thin and watery, more or less translucent, and shows a distinct yellowish 
color. 










^ 




{ 


^ ^ 






o 


^ 


'S' 


b- 






Fig. i6o. — Normal Milk and Colostrum. (Hawk.) 
a, Normal milk; b, colostrum. 

Microscopic examination shows the presence of rather large cells in which 
are many fat granules and occasionally a distinct nucleus. This secretion, 
which is called colostrum,^ continues for three to four days and is distinguish- 
able from the later secretion by the fact that it contains relatively more salts 
than normal milk and, according to the usual statements, more sugar. In a 
series of determinations of breast milk collected during the fourth day after 

^ See Engel and Bauer, Die Biochemie und Biologic des Kolostrums, Wiesbaden, 1912, 
Bergmann; Benestad, Montasschr. f. Geburtsh. u. Gynak., 1914, XL, 674; Holt, Courtney 
and Fales (Am. Jour. Dis. Child., 1915, X, 229) indicate that in the colostrum period, 
woman's milk has high protein and high ash with rather low fat values; Hammett(Jour. 
Biol. Chem., 1917, XXIX, 381) shows that fat and lactose were increased in amount and 
protein diminished during the first eleven days after parturition. 

766 



SECRETION OF THE MAMMARY GLANDS 767 

delivery, the writer could not show any marked increase in the sugar in all 
cases, but in three out of eight cases examined a percentage higher than eight 
of lactose was obtained. 

It is probable that the function of the colostrum is gradually to accustom 
the child to the taking of food by diminishing to a slight extent the elements, 
protein and fat, which are more apt to cause digestive disturbances than is the 
lactose. 

The secretion of true milk begins about the fourth day and continues for 
a variable length of time. Marked variations are observed both in the quan- 
tity and the quality of this secretion in various women so that no hard and 
fast rule can be given as to the composition of normal mother's milk. While 
it is probably true that the normal woman should nurse her child during a large 
part of the first year, it is rare to find, especially in private practice, many 
such cases. Either the milk becomes scanty and loses in nutritive power or 
becomes excessive and consequently diluted. In either case the child is not 
receiving the most suitable nourishment, so that breast-feeding is abandoned 
under these circumstances. It seems to be a general rule, which is difficultly 
explicable, that the more highly socially developed the woman the less apt she 
is to nurse her child with any success. It is possible, and this seems to the 
writer the most probable explanation, that the child usually receives more or 
less constant attention from the physician, and variations in the breast milk 
are more frequently noticed than in the case of the poorer child who rarely 
has the advantage of medical attention unless more or less serious illness 
occurs. 

II. Physical and Chemical Properties 

Various figures have been given for the composition of human milk so that 
it is difficult to strike an average. So much depends upon the nourishment of 
the mother, upon the amount of exercise taken by her, and upon the general 
condition of the system that marked variations exist in the proportion of the 
chemical constituents. The following table, taking averages of examinations 
reported by various writers, may serve as one representing more nearly the 
normal condition.^ 



Water, 

Salts, 

Protein, 


87.24 
0.26 
1.50 


Fat, 


4.00 



Lactose, 7.00 

These figures are not as high as regards protein as those usually given, but 
are higher as far as the lactose is concerned. Although many writers have 

^ See Schloss, Monatsschr. f. Kinderhkde., 191 2, X, 499; also, Meigs and Marsh, Jour. 
Biol. Chem., 1913, XVI, 147; Talbot, Am. Jour. Dis. Child., 1914, VII, 445; Grulee and 
Caldwell, Ibid., 1915, IX, 374; Hart and Humphrey, Jour. Biol. Chem., i9i5,XXI, 239; 
Bosworth and Van Slyke, Jour. Biol. Chem., 1916, XXIV, 187; Hammett and McNeile, 
Ibid., 191 7, XXX, 145; Raimondi, Le Nourrison, 1919, IV, 269. 



768 



DIAGNOSTIC METHODS 



I ' 



M 



given the percentage of protein much higher than 1.5 per cent., the writer has 
never been able, in several hundred examinations of milk in his laboratory, to 
find many showing a protein percentage of two or more. 

As it is frequently necessary to modify cow's milk so that it may more 
nearly approach mother's milk in composition, the writer inserts the following 
table for comparison. 



Water, 


87.25 


Salts, 


0.75 


Protein, 


3-50 


Fat, 


4.00 


Lactose, 


4-50 



It will be seen that cow's milk shows a higher per cent, of protein and a 
lower per cent, of carbohydrate. It is necessary, therefore, that this be modi- 
fied by diluting the milk so as to diminish the protein and by adding lactose to 
make up for the deficiency of carbohydrate. In the dilution the fat content 
will necessarily be lowered so that this may be remedied, as suggested by 
Backhaus, by the addition of cream. The writer must refer to works on 
pediatrics for the various methods of modifying cow's milk. It is to be re- 
membered that no modification is equal to mother's milk The casein of 
human milk forms a much finer clot with the gastric juice than does that of 
cow's milk so that the latter may not be well tolerated by the child. More- 
over, some unknown principle present in human milk is accountable for a 
distinct biologic difference in these two types of nutritive material. 

(i) Appearance and Color. 

Normal human milk is a white fluid which usually has a slight bluish tinge 
except immediately after birth when the color may be distinctly yellowish 
from the presence of colostrum.^ If the percentage of fat be relatively high 
the color will be more nearly a pure white with little of the blue tone. 

(2) Specific Gravity. 

The specific gravity of human milk varies between 1,028 and 1,034. An 
increase in the percentage of fat will usually lower the specific gravity while 
the protein and carbohydrates will increase it. 

Cow's milk shows approximately the same specific gravity. If a low spe- 
cific gravity is obtained it is evidence either of an increased percentage of fat 
or of dilution with water. If the percentage of fat be low the milk is un- 
questionably a watered one. If the milk tested shows a high specific gravity 
the chances are that most of the fat has been removed either by skimming or 
by centrifugation. 

The determination of the specific gravity may be made by a special in- 
strument known as Quevenne's lactodensimeter or by the use of the ordinary 
hydrometer used in urine work. As it is never of any clinical importance or 
even of any marked practical importance that the specific gravity should be 
absolutely accurately determined, the writer is accustomed to use the urino- 

^ Feer (Biochem. Ztschr., 1916, LXXII, 378) has reported a greenish coloration of the 
milk after eating calves liver. 



SECRETION OF THE MAMMARY GLANDS 769 

meter for such determinations. Corrections for variations in temperature 
are necessary only when great differences exist between the temperature of 
the room and the temperature at which the instrument is calibrated. 

(3) Reaction. 

Normal human milk as well as cow's milk shows an amphoteric reaction 
to htmus-paper and an acid reaction to phenolphthalein, cow's milk being 
somewhat more acid than mother's milk toward the latter indicator.^ 

(4) Coagulation. 

If milk be allowed to stand, the reaction gradually becomes more and 
more acid owing to the development of bacteria, especially of the bacillus 
acidi lactici. When the degree of acidity reaches a certain point, casein sepa- 
rates first in the form of flocculi and later the entire fluid may coagulate to 
a jelly-like mass. This mass soon contracts and settles out leaving a slightly 
turbid fluid known as milk plasma or acid whey.^ In order to inhibit the 
development of bacteria and prevent this coagulation, known as souring, 
certain preservatives are frequently added to market milk and should be 
capable of detection by the practitioner. These will be discussed in a later 
section. 

Besides this type of coagulation of cow's milk a second form is observed 
which takes place under the influence of chymosin without any change in the 
reaction of the fluid. In this case the whey is sweet and contains practically 
all of the lactose originally present in the milk.^ 

(5) Total Solids. 

Five to ten c.c. of the well-mixed milk are placed in a weighed platinum 
dish, evaporated to dryness on a water-bath, and dried to constant weight 
in the oven at 105°. The difference in weight between the platinum dish 
and its contents, on the one hand, and the platinum dish, on the other, gives 
the amount of total solids in the milk taken. A simple calculation will yield 
the percentage of total soHds. 

Under normal conditions the total solids of both human and cow's milk 
should average between 12 and 13 per cent. Variations in this figure are, 
of course, due to fluctuations in the various constituent elements. 

(6) Ash. 

The platinum dish containing the dried residue of the milk is heated 
over a direct flame until the residue is completely incinerated. This is then 
placed in the desiccator and dried to constant weight. The difference in 
weight between the dish and contents and the dish alone represents the 
amount of salts present in the milk originally taken. '^ 

1 See Clark, Jour. Med. Research, 1914, XXXI, 431, who shows that the Ph vakie for 
mother's milk is 7 to 7.2; Milroy, Biochem. Jour., 191 5, IX, 215; Van Slyke and Baker, 
Jour. Biol. Chem., 1919, XL, 345 and 357. 

^ See Orla-Jensen, Ztschr. f. physiol. Chem., 1914, XCIII. 283; Van Slyke and Bos- 
worth, Jour. Biol. Chem., 1916, XXIV, 191; Van Slyke and Baker, Ibid., 1918, XXXV, 
147- Sommer and Hart, Ibid., 313. 

* See Sommer and Hart (Jour. Biol. Chem., 1919, XL, 137) for a discussion of heat 
coagulation of milk. 

^ Bos worth. Jour. Biol. Chem., 191 5, XX, 707; Holt, Courtney and Fales, Am. Jour Dis. 
Child., 1915, X, 229; Meigs, Blatherwick and Cary, Jour. Biol. Chem., 1919, XXXVII, i. 

49 



il 





4 grains 


nt.), 


8 ex. 


, qs., ad., 


200 c.c. 



770 DIAGNOSTIC METHODS 

(7) Protein. 

The methods for the determination of protein are divided into those for 
estimation of the total protein material present and into those in which sepa- 
rate determinations are made of the casein, on the one hand, and albumin 
and globulin, on the other. 

{a) Total Protein. 
Method of Sebelien. 

Ten c.c. of milk are diluted with 90 c.c. of water, 5 c.c. of a saturated so- 
dium chlorid solution, and 15 c.c. of Almen's tannic acid solution are added. 
The mixture is thoroughly stirred and the dense precipitate which forms is 
allowed to settle. The composition of Almen's tannic acid solution is as 
follows: 

Tannic acid. 

Acetic acid (25 per cent.). 

Alcohol (50 per cent. 

The precipitate, which consists of the total protein of the milk and a large 
portion of the fat carried down by the precipitate, is then filtered off through 
a fine filter and is washed with cold water. The filter-paper and its contents 
are then placed in a Kjeldahl flask and a nitrogen determination made as 
described under Urine. It is advisable to use 20 c.c. of sulphuric acid instead 
of the ten employed in the case of urine, as the mixture oxidizes much more 
rapidly under these conditions. If the nitrogen obtained in this determina- 
tion be multiplied by 6.37, the result will be the protein in 10 c.c. of milk. 

Method of Boggs. 

For routine work this method is perhaps more advisable for the general 
practitioner than is the preceding, but it does not always give confirmatory re- 
sults. It is based upon the fact that the total protein of milk is precipitated by 
phosphotungstic acid in hydrochloric acid solution, the amount of precipitate 
being measured in an Esbach tube. The reagent used has the following 
composition: 

Phosphotungstic acid, 25 grams. 

Concentrated hydrochloric acid, 25 c.c. 

Distilled water, q.s., ad., 250 c.c. 

It has been found that the milk should be diluted with water before adding 
the reagent if the results are to be accurate. As a rule, a dilution of i to 10 
for human milk and i to 20 for cow's milk suffices. 

The diluted milk is poured into the Esbach tube^ to the mark U and 
the reagent added to the mark R. The tube is then closed with a stopper 
and inverted several times thoroughly to mix the contents. It is then set 

^ If a Purdy centrifuge tube be filled with the diluted milk to the 10 c.c. mark and the 
reagent added to 15 c.c, the amount of protein may be determined quickly by centrifuging 
for 3 minutes and reading the percentage directly. The figures obtained agree quite closely 
with those of the gravimetric method. 



SECRETION OE THE MAMMARY GLANDS 771 

aside for 24 hours when the percentage of protein in the milk is read off di- 
rectly from the calibrations on the tube in case the dilution was i to 10, 
while with a dilution of i to 20 the figures are multiplied by 2. 

This method, while convenient, is open to the objection that many factors 
may influence the depth to which a precipitate settles. Moreover, the 
Esbach tubes reading as high as 12 parts do not give as satisfactory results as 
those with readings from one to seven. Such being the case the method must 
be used more for clinical purposes than for scientific estimations. 

(b) Casein. 

Twenty c.c. of well-mixed milk are measured into a beaker and approxi- 
mately 380 c.c. of water are added. The mixture is thoroughly stirred and 
very dilute acetic acid added drop by drop with constant stirring until a floc- 
culent precipitate is observed. When this point is reached a stream of car- 
bon dioxid is passed through the mixture for one-half hour, after which the 
-vessel is allowed to stand until the next day. The above part of the technic 
is directly applicable to cow's milk. If human milk is being examined it is 
necessary to heat the vessel to 4o°C. during the passage of the carbon dioxid. 

After the mixture has stood overnight, it is filtered through a nitrogen- 
free filter and washed with water. The residue on the filter contains casein 
which is mixed with a portion of the fat present. The filter-paper and con- 
tents are then placed in a Kjeldahl flask and a nitrogen determination is made 
as previously described. Multiplication of the nitrogen value by 6.37 yields 
the amount of casein in the 20 c.c. of milk originally taken. ^ 

(c) Albumin and Globulin. 

The filtrate from the above precipitation of casein contains the remainder 
of the protein material and the carbohydrate of the mflk. This filtrate is 
placed in a porcelain dish and heated for a few minutes to the boiling-point. 
The protein material is coagulated and may be filtered through a nitrogen- 
free filter and washed several times with cold water. A nitrogen determina- 
tion is then made and the value multiplied by 6.37 to obtain the amount of 
albumin and globulin present in the 20 c.c. of milk.^ 

The filtrate from this latter precipitation contains the lactose, which may 
then be determined by titration with Fehling's solution, as described under 
Urine. It is to be remembered that 10 c.c. of Fehling's solution are reduced 
by 0.0678 gram of lactose, and not by 0.05 as in the case of glucose. 

^ See Van Slyke and Bosworth, Jour. Biol. Chem., 1915, XX, 135; Hart and Humph- 
rey, Jour. Biol. Chem., 191 7, XXXI, 445; Osborne and Wakeman; Ibid., 19 18, XXXIII, 
7; Bosworth and GibHn, Ibid., XXXV, 115; Hart and Humphrey, Ibid., 367; Palmer and 
Scott, Ibid., 1919, XXXVII, 271; Van Slyke and Bosworth, Ibid., 285. 

2 See Emmett and Luros, Jour. Biol. Chem., 1919, XXXVIII, 257. Denis and Minot 
(Ibid., XXXVII, 353 and XXXVIII, 453) have introduced methods for the determination 
of the non-protein nitrogenous constituents of milk. (See, also, Kennedy, Jour. Am. 
Chem. Soc, 1919, XLI, 388; and Denis, Talbot, and Minot, Jour. Biol. Chem., 1919, 
XXXIX, 47). Osborne and Wakeman (Ibid., 1918, XXXIII, 243) have sliown the 
presence of a further protein in cow's milk which is soluble in 50 to 70 per cent, alcohol 
but insoluble in absolute alcohol. They point out that this must be taken into consid- 
eration in methods having to do with the estimation of the non-protein nitrogenous 
constituents. 



772 



DIAGNOSTIC METHODS 



(8) Fat. 

It is important in the determination of the fat content of milk that a 
thoroughly mixed specimen be examined.^ As the fat tends to rise to the 
surface of the milk, the fluid should be poured from one vessel into another 
several times to insure thorough mixing and an immediate measurement 
made of the portion to be tested. 

For clinical purposes as well as for examination of market milk the method 
of Babcock is to be recommended. For accurate results, however, this 
method is not to be advised. 



flM 



iC.C. 



Bab cock's Method. 

This method consists in the destruction of the organic matter, except 
the fat, by means of sulphuric acid. The fat is then separated by centrifug- 
ing and determined by reading off the percentage from 
the calibrations in the neck of the bottle used. 

In the case of cow's milk or where sufficient human 
milk may be obtained, 17.6 c.c. of milk are measured into 
the bottle and 17.5 c.c. of sulphuric acid added. These 
fluids are then mixed by shaking and rotating the bottle 
in such a way that no curds pass into the neck of the 
bottle. As soon as the mixture becomes homogeneous and 
dark brown or even black in color, the bottles are placed 
in a special centrifugal machine and whirled for five minutes. 

If the room be very cold it is 
advisable to fill the holders of the 
centrifuge with boiling water in 
order to keep the fat melted 
while it is being centrifuged. 
At the end of five minutes, centri- 
fugation is discontinued and the 
neck of the bottle filled with 
boiling water. The melted fat 
will rise in the neck of the flask 
and may be read off from the 
calibration. In order to facih- 
tate this the bottles are again 
centrifuged for one minute. 

If a small amount of milk 
only is 

tubes shown in cut may be 
used. Milk is added to the mark five and sulphuric acid poured in so as 
to fill the body of the tube. It is usually necessary to add the milk and 
acid by means of thin narrow pipets, as the neck of the tube is too small 
to" permit of easy entrance of the fluids otherwise. The milk and sul- 

1 See Brodrick-Pittard, Biochem. Zt^.chr., 19 14, LXVII, 382; Osborne and Wakeman, 
Biol. Chem., 1915, XXI, 539; Ibid., 1916, XXVIII, i; Denis and Minot, Ibid., 1918, 
XXXVI, 59. 



Fig. 161. — Babcock Bottles. 
a, Milk bottle; 6, cream bottle 



Bottle 



available the smafler ^l^; ^^^^^^ ^^^^ 



SECRETION OF THE MAMMARY GLANDS 773 

phuric acid are mixed by rotation of the tube until a homogeneous fluid 
results. The mixture is centrifuged for a few minutes, the neck of the 
tube being filled with a mixture consisting of equal parts of concentrated 
hydrochloric acid and amyl alcohol. The percentage of fat is then read 
off from the calibrations on the tube. 

Extraction Method. 

A few grams of dried washed sand are placed in the extraction shell of a 
Soxhlet apparatus and lo c.c. of well-mixed milk are allowed to fall upon 
it drop by drop. This is dried at a temperature of ioo°C. for one to two 
hours and is placed in the tube of the extraction apparatus. The fat is 
extracted in the usual way by the use of gasoline or anhydrous ether, com- 
plete extraction usually requiring from two to three hours. The apparatus 
is disconnected, the ether evaporated from the distilling flask, the residue 
in the flask dried at ioo°C., and the flask and contents dried to constant 
weight in the desiccator. The difference between the original weight of the 
flask and its weight including the extracted residue, yields the amount of fat 
in the lo c.c. of milk. This method is the most accurate one, but is not as 
convenient as the preceding for the general practitioner. 

(9) Lactose. 

In general routine analyses of milk the lactose may be determined by 
difference. By this is meant that subtraction of the values for the sum of 
water, ash, protein, and fat from 100 will yield the pertentage of lactose. 
For clinical purposes this is usually sufflcient, but for the more accurate 
work it does not give exact figures, as shght amounts of other undeter- 
mined substances are present. 

For most direct determinations of lactose it is necessary that the larger 
portion of the protein material be removed previously. This may be done 
by the method outlined under Determination of Casein, Albumin, and 
Globulin; or for clinical purposes sufficiently accurate results may be reached 
by acidifying with acetic acid, boiling, and filtering. It is advisable always 
to take the time to saturate the mixture with carbon dioxid after the casein 
has been precipitated with acetic acid, as the results are more satisfactory.^ 

The fluid is then titrated by the use of Bang's or Purdy's solution, using 
all the precautions mentioned under these tests in the section on Urine. 
The 35 c.c. of Purdy's solution are reduced by 0.02712 gram of lactose. 

Folin and Denis^ have applied the method of Folin and McEllroy to the 
determination of the lactose in milk. In a later communication Folin and 
Peck modified this latter method to a slight extent, as certain difficulties 
had been met with in the preparation of the salt mixture used in the test. 
This method is a very exact one and is especially recommended for the deter- 
mination of lactose in milk as it is unnecessary to precipitate the protein 

^ See Jackson and Rothera, Biochem. Jour., 1914, VIII, i; Palmegiani, Pediatria, 1914, 
XXII, 739; Hill, Jour. Biol. Chem., 1915, XX, 175. 

-Jour. Biol. Chem., 1918, XXXIII, 521. See, also, Pacini and Russell, Ibid., 1918, 
XXXIV, 505; Folin, Denis, and Minot, Ibid., 1919, XXXVII, 349; Talbot, Jour. A. M. A., 
1919, LXXIII, 138; Denis and Talbot, Am. Jour. Dis. Child., 1919, XVIII, 93. 



774 



DIAGNOSTIC METHODS 



^ 



before making the titration. It has been discussed in detail on page 312, 
but certain points, applying to milk, must be mentioned here. The milk 
must be diluted with water before making the determination. A dilution of 
1 :4 for cow's milk and 1 15 for mother's milk are satisfactory. The titration is 
made as follows: Into a large test-tube introduce 2.8 to 3.4 c.c. of the diluted 

milk (that is, nearly enough to produce 
complete reduction of the copper solution — 
5 c.c. of the copper solution, a pebble (to 
prevent bumping), and 4 or 5 grams of the 
salt mixture previously mentioned. Shake 
weU and boil gently for 4 minutes before 
adding any more milk. At the end of this 
time add more milk (0.02 to o.i c.c, depend- 
ing on the amount of blue color remaining) 
and boil for i minute. The boiling should 
be for I minute after each addition of milk, 
the total boiling period being 5 to 7 minutes. 
The end reaction is determined by the de- 
coloration of the solution and the precipita- 
tion of the white cuprous thiocyanate. In 
this test the amount of copper solution used 
is decolorized by 40.4 mg. of anhydrous 
lactose. Hence, 4.04 multiplied by the 
degree of dilution (4 or 5) and divided by 
the titration figure gives the per cent, of 
lactose present in the milk. 

If the polarimeter is to be used for the 
estimation of lactose in milk the following 
procedure may be used. Fifty c.c. of the well- 
mixed milk are placed in a flask, 25 c.c. of a 
solution of neutral lead acetate are added, 
the flask is closed with a stopper through 
which passes' a glass tube approximately 30 
cm. in length. The mixture is then heated 
over a small flame to boiling. After the mixture has cooled it is filtered 
through a dry filter into a dry vessel and polarized. 




Fig. 



163. — Soxhlet apparatus. 
(Hawk.) 



(10) Preservatives in Cow's Milk. 
(a) Sodium Carbonate. 

To hide the acid reaction of a spoiled sample of milk sodium carbonate 
is often added, and may be detected as follows: Ten c.c. of milk are mixed 
with ten c.c. of 96 per cent, alcohol and a drop of rosolic acid solution. Pure 
unadulterated milk produces a brownish-yellow color, but, in the presence 
of sodium carbonate or bicarbonate, a rose color is obtained. For greater 
precision in doubtful cases the questionable sample should be compared with 
known unadulterated milk. Phenol-phthalein solution may be used as an in- 



SECRETION OF THE MAMMARY GLANDS 77 5 

dicator in place of rosolic acid. By this method 0.05 per cent, of carbonates 
may easily be detected. 

(b) Salicylic Acid. 

Twenty ex. of milk are treated with two or three drops of sulphuric acid 
and shaken with an equal amount of ether. The greatest possible part of the 
ethereal solution is drawn off and evaporated, the residue extracted with 
40 per cent, alcohol, filtered, and 5 ex. of the filtrate treated with a few drops 
of ferric chlorid solution. A violet color shows the presence of salicylic 
acid or some other hydroxy derivative of benzol. 

(c) Formaldehyd. 

Two c.c. of concentrated sulphuric acid are placed in a test-tube and a 
drop of ferric chlorid solution added. A few c.c. of milk are allowed to run 
from a pipet upon the surface of the mixture in such a way that a distinct 
line of contact forms. A violet color at the point of contact of the two liquids 
is characteristic of formaldehyd in the presence of casein. 

(d) Boric Acid and Borax. 

Fifty c.c. of milk are alkaHnized with milk of lime, evaporated to dryness 
and incinerated. The resulting white crystalline residue is treated with a 
few drops of tincture of turmeric and very dilute hydrochloric acid and is 
then dried on the water bath. The presence of the shghtest trace of boric 
acid gives to the dry residue a beautiful vermilion or cherry red color. It is 
possible by this method to detect o.ooi per cent, of boric acid in milk. Only 
very dilute hydrochloric acid must be used in testing for boric acid, since the 
concentrated acid itself gives with tincture of turmeric a red color. The 
coloration produced by boric acid is distinguished from that produced by 
hydrochloric acid by the fact that it does not disappear by treatment with 
water in the cold, but only after long boihng, while the color caused by hydro- 
chloric acid disappears as soon as it is diluted with water. 

If the crystalline residue obtained as above described be treated with 
alcohol and the alcohol ignited, a flame tinged with a beautiful emerald-green 
color is obtained in the presence of boric acid. 

III. Bacteriological Examination of Milk 

The bacteriological examination of human milk is frequently desirable 
from a clinical standpoint as milk may become contaminated as it passes 
along the lacteal ducts. In pathologic conditions many types of organisms, 
such as typhoid bacilli, pneumococci, and tubercle bacilli may be obtained, 
although the latter are extremely rare. 

With cow's milk the chief question at issue is whether the milk contains 
sufficient bacteria to be harmful to the child. Aside from the presence of the 
tubercle bacillus in the milk of infected animals, large numbers of saprophytic 
organisms must find their way into this fluid and will, if in large numbers, 
influence the intestinal activity of the child. Moreover, it must be remem- 



776 



DIAGNOSTIC METHODS 



bered that many epidemics of milk-borne disease, such as typhoid fever 
and streptococcic sore-throat, may arise owing to improper handhng of the 
milk or to inefficient pasteurization.^ 

The methods of examining milk for bacteriologic differentiation are the 
same as for any other fluid and will be discussed in the next chapter. It is, 
not infrequently, a matter of importance to the general practitioner and of 
untold value to health officers to determine the number of bacteria in the milk 
supplied to the community. The outline of the method to be followed in this 
work is as follows: 

Collect the specimen as freshly as possible; Keep it on ice from the time 
of collection until you are ready for the examination. Sterilize a quantity of 
distilled water in the autoclave. Plug a number of pipets (i and lo c.c.) with 
cotton and sterilize in the hot oven. A number of Erlenmeyer flasks, plugged 
with cotton, a loo c.c. graduate,' and a number of Petri dishes are sterilized at 
the same time. 




Fig. 164. — Wolfhiigel's colony counter. 



'i 



11/ 



By means of the graduate place 99 c.c. of sterile distilled water in an Erlen- 
meyer flask, 90 c.c. in a second and 99 c.c. in a third and replace the cotton. 
By means of a sterile i c.c. pipet transfer i c.c. of the well-shaken specimen 
to flask number i. The dilution of the milk in this first flask is, therefore, i 
to 100. Shake this dilution thoroughly and transfer 10 c.c. of this dilution, by 
means of a pipet, to the second flask containing 90 c.c. of water. The second 
dilution is i to 1,000. By means of a second i c.c. pipet transfer i c.c. of the i 
to 100 dilution to the last flask, containing 99 c.c. of water, thus obtaining a 
dilution of i to 10,000. These dilutions are usually all that are necessary. 
Others may be made as desired. In the examination of the best market milks, 
the I to 100 dilution is sufficient. ^ 

Liquefy a number of tubes of litmus-lactose agar, litmus-lactose-bile agar 
and plain agar and cool them down to 40° to 45°C. Transfer i c.c. of each 
dilution of the milk to each of two sterile Petri dishes and add the liquefied 
culture medium. Mix by careful manipulation and allow the media to harden. 
Place in incubator for 48 hours and count the number of colonies, using a 
colony counter such as shown in the illustration. Multiply the number of 

^ See discussion of "Streptococcic Sore Throat" on page 41; also Rogers and Dahlberg, 
Jour. Agricul. Research, 1914, I, 491. Shippen (Jour. Am. Med. Assn., 1915, LXIV, 1289) 
shows, in conformation of the work of De Jong and De Graef, that colon bacilli may sur- 
vive the pasteurization process and are, therefore, not to be taken as evidence of later 
contamination. 

2 See Wells, Am. Jour. Pub. Health, 1919, IX, 956, for a discussion of standard method 
for bacteriological dilution. 



SECRETION OF THE MAMMARY GLANDS 777 

colonies by the dilution to obtain the actual number of colonies per c.c. of 
original milk.^ 

The number of bacteria in milk may vary from a few thousand to many 
millions. It is an impossibility to obtain a specimen which is sterile so that 
the presence of as high as 100,000 organisms should not be regarded as danger- 
ous unless pathogenic types are present. If the freshly examined specimen 
contains between 10,000 and 50,000 bacteria per c.c, it is probable that all 
possible precautions have been taken to prevent contamination, although the 
writer has found some specimens showing a count of only 100 per c.c. 

In the examination of milk it is customary also to make a cytological 
examination. It was formerly supposed that normal fresh milk contained 
only a few scattered leucocytes and epithelial cells, any marked increase in 
their number being taken as evidence of infection. However, by use of more 
careful methods it has been shown that the number of these cells may run as 
high as 500,000 or more and yet no abnormality be present. It is necessary 
in this work to distinguish between the ordinary leucocytes, which are con- 
stantly discharged in large numbers in the milk, and the true pus cells, which 
are evidence of disease. No detailed work along this line has been reported. 
The association of large numbers of bacteria, especially of staphylococci and 
streptococci, is probably evidence of infection of the animal and consequent 
unfitness of the milk for use, although the significance of the increase in 
cellular elements is not, by any means, settled. The methods used in this 
cytological examination are the same as outlined previously. ^ 

BIBLIOGRAPHY 

1. CzERNY UND Keller. Die Nahrungs-Pathologie. Berlin, 1907. 

2. Holt. Diseases of Infancy and Childhood. New York, 1906. 

3. Langstein and Meyer. Sauglingsernahrung und Sauglingsstoffwechsel. Wies- 

baden, 1914. 

^ See Brew, New York Agricultural Exper. Station, Geneva, N. Y., Bull. 373, 1914; 
also, Goodrich, Jour. Infect. Dis., 1914, XIV, 512. 

2 See Lewis, Am. Jour. Dis. Child., 1913, VI, 225; Hachtel, Jour. Am. Med. Assn., 1913, 
XI, 565; Stokes and Stoner, Ibid., 1024; Breed, Bull. 380, New York Agricultural Exp. 
Station, Geneva, N. Y., 1914; Hewlett and Revis, Lancet, 191 5, I, 855; Frost, Jour. Am. 
Med. Assn., 1915, LX, 821; Ibid., 1916, LXVI, 889; Levine and Emerson, Jour. Infect. 
Dis., 1916, XVIII, 143; Evans, Ibid., 437; Davis, Ibid., XIX, 236; Frost, Ibid., 273; Allen, 
Ibid., 712; Jordan, jour. A. M. A., 1917, LXVIII, loSo; Simmons, Jour. Infect. Dis., 
1919, XXIV, 322. 



CHAPTER XI 



CLINICAL BACTERIOLOGY 




I. General Considerations 

In no field of medical or other scientific research have there been recent 
advances of such far-reaching importance to humanity as in the realm of 
Clinical Bacteriology. The study of the relationship of bacteria and their 
metabolic products to disease in general and the identification of the specific 
organism of many of our most dreaded diseases has led to such an enormous 
output of work upon the various ways in which the system may react to bac- 
terial invasion, that our knowledge of the subject of immunity, both natural 
and acquired as well as active and passive, has advanced with leaps and 




Fig. 165. — ^Lautenschlager Hot-air Sterilizer. 

bounds. Indeed, so greatly has our knowledge increased that preventive 
medicine is fast taking the place of curative medicine. It is evident, there- 
fore, that the scientific aspect of bacteriology, great as it is, is, for the general 
worker and for the public at large, overshadowed by the success attached to 
the practical application of the knowledge acquired from the hard and earnest 
work of scores of scientific investigators. Scientific and applied bacteriology 
must go hand in hand, each playing its important part in the struggle against 
disease. 

The object of the writer in presenting this section is not to include a dis- 
cussion of the advances in bacteriology, as this would, indeed, be absurd. He 
does wish, however, to present certain general features of the subject, so that 

778 



CLINICAL BACTERIOLOGY 



779 



the student and practitioner may be given- such a working knowledge as will 
enable them properly to perform and interpret many of the tests, which they 
may have occasion to undertake. Many such points have been discussed 
throughout this book but many have been omitted, so that the writer feels that 
this discussion will not be untimely. 

II. Sterilization 

As is well known, bacteria are practically 
ubiquitous, being found in air, water, food and 
soil. It is evident, therefore, that all material 
and glass-ware, which may be used in bacterio- 
logical work, must be clean and sterile before 
any reliable results may be obtained. 

Glass-ware should be thoroughly cleaned 
by boihng in soapsuds or by soaking for a 
few hours in the following cleaning mixture. 

Potassium bichromate, 60 grams. 

Water, 300 c.c. 

Concentrated sulphuric acid, 460 c.c. 

After washing or soaking, rinse the glass 
thoroughly in running water and dry. Plug 
the test-tubes and flasks with non-absorbent 
cotton and place in a hot-air sterilizer or 
oven for one hour at a temperature of i7o°C. 
The internal temperature of the sterilizer must 
be carefully regulated or the variation may be 
so great in different portions of the sterilizer 
that the more resistant organisms and spores 
will not be kiUed. 

Culture media are best steriHzed by the use 
of the autoclave, an apparatus for holding 
steam under pressure. The usual pressure 
used is 15 pounds, the media being kept in con- 
tact with steam, at this pressure (temperature 
about i2o°C.) for 15 to 20 minutes. The gas 

is then turned off, and the apparatus allowed to cool. While most media may 
be sterilized by this method, gelatin media would better not be heated longer 
than five minutes and blood-serum and carbohydrate-containing media may 
undergo chemical change if the heating is prolonged. 

For this reason, such media as last mentioned or all media, in the absence 
of an autoclave, may be sterilized in the Arnold sterilizer, an apparatus so con- 
structed as to produce steam from a small amount of water, the tank being 
fed by the water of condensation. As many bacterial spores are not killed by 
live steam, it is necessary to use the so-called discontinuous sterilization when 




Fig. 166. — Autoclave. 



78o 



DIAGNOSTIC METHODS 



the Arnold is employed. Heat the media for 30 minutes after the steam has 
filled the sterilizer and allow the media to stand at room temperature for 24 
hours. Repeat the process on each of two other successive days. In this way 
spores develop into vegetative organisms, which are killed by the later heating. 
Certain fluids, such as serum or ascitic fluid, cannot be sterilized by heat- 
ing owing to coagulation. These are sterilized by passing through a Pasteur, 
Chamberland or Berkefeld filter. 



dtii 




Arnold Sterilizer. 



III. Preparation of Culture Media 

For the development of bacteria certain factors are essential. The most 
important of these are (i) oxygen; (2) food; (3) moisture; (4) a proper reaction 
of the culture media; and (5) an optimum temperature for growth. 

Regarding the question of proper food it is to be said that the number of 
culture media advocated is legion.^ I cannot, therefore, attempt to give any 
but those in general use in laboratory routine. Many special media have 
been discussed in other sections of this book and will be referred to later in the 
discussion of special organisms. 

Nutrient Broth (Bouillon ; Beef Broth). 

Infuse 500 grams of chopped lean meat with 1,000 c.c. of distilled water 
for 24 hours in the ice box. Instead of this meat, one may use 3 grams of 
Liebig's meat extract, but the medium is not as nutritious. Strain the infu- 

^ While it is true, as Rettger and his associates have shown, that purified unaltered 
proteins are resistant to bacterial attack, in the case of the simpler nitrogenous compounds 
we find that bacteria synthesize their own protoplasm when grown in synthetic media 
containing amino acids, or even ammonium phosphate or uric acid. See, Koser and Rett- 
ger, Jour. Infect. Dis., 1919, XXIV, 301. 



CLINICAL BACTERIOLOGY 78 1 

sion through cotton flannel, the juice being well pressed out. Add i per cent, 
peptone (lo grams). Warm on water bath, stirring until the peptone is 
dissolved. Heat over boiling water or steam for 30 minutes. Restore the 
original volume of fluid. Titrate the acidity of the broth, using N/20 sodium 
hydrate solution and phenolphthalein as an indicator.^ Adjust the reaction 

^ The optimum reaction for such culture medium is one having a neutral reaction to 
litmus and a degree of acidity requiring i c.c. of normal (4 per cent.) sodium hydrate to 
neutralize 100 c.c. of the medium. This is known as +1. See Clark, Jour. Infect. Dis., 
191S, XVII, 109. 

Place 5 c.c. of the medium to be tested in a porcelain dish and add 45 c.c. of distilled 
water. Boil to expel carbon- dioxid. Add i c.c. of 3"^ per cent, solution of phenolphthalein 
in 50 per cent, alcohol. Titrate, while hot, with N/20 sodium hydrate up to a faint but 
distinct pink color. Read from the buret the amount of N/20 NaOH used to neutralize the 
5 c.c. of medium and calculate the amount of normal NaOH necessary to neutrahze the 
entire quantity of bouillon. 

Thus, if 1.5 c.c. of N/20 NaOH were used for 5 c.c, this would represent 30 c.c. for 100 
or 300 for 1,000 c.c. But 300 c.c of N/ 20 NaOH is equivalent to 15 c.c. of normal NaOH. 
As we desire the medium to have an end-reaction of +1 per hundred or 10 per liter, we must 
evidently add 5 c.c. of normal NaOH to bring this medium up to the proper standard. 

While the above method is still used by some workers, it is more than probable that, 
ere long, this system will give way, entirely, as it has done in many laboratories, to the more 
reliable and scientific method of adjustment of culture media in terms of its Ph values. 
This method is discussed in great detail under Blood, but a few remarks, specially directed 
toward the application of this work to culture media are necessary. As Clark and Lubs 
have pointed out, the reference point in determining titratable acidities of culture media 
is, in reality, but a Ph point indicated by the tint of phenol phthalein. The particular 
tint adopted by different workers varies, so that the tint which is sometimes assumed is 
that at which phenol phthalein is least sensitive. Without a proper standard comparison 
solution of known Ph value the same worker can hardly avoid errors of o.i Ph in establish- 
ing his reference point for titratable acidity. The colorimetric method of adjusting the 
reaction of culture media on the basis of its Ph value is a return to the older method of ad- 
justing to a given tint of an indicator but with more reliable indicators, a wider choice of 
indicators, a logical scale of reaction, and a clearer conception of the ends desired in the 
adjustment. The method of procedure is as follows: A measured 10 c.c. of the broth, whose 
reaction is to be adjusted, are placed in a clean test-tube which has been rinsed with a por- 
tion of the broth. To this is added J^-^ c.c. of a 0.02 per cent, solution of phenol-sul- 
phonephthalein (phenol red). Add, from a buret, a measured quantity of N/20 sodium 
hydrate solution, until the color of the mixture matches that of the standard solution, 
whose Ph value equals that chosen for the culture media. This comparison of color 
tints is made in a comparator, which consists of a simple wooden box with holes in the top 
for the test tubes and J'i inch holes front and back to permit the passage of light through 
the solutions placed in the tubes. (While, for the usual adjustments of reaction of culture 
media, phenol red is satisfactory, yet cases may arise in which the range of Ph values 
indicated by this indicator does not fall within the scope of this indicator. In such cases 
other indicators, as mentioned under Blood must be used.) A simple calculation suffices 
to determine how much N/i NaOH must be added to the remainder of the broth to bring 
the entire amount to the reaction of the chosen standard. Liquid media may be titrated 
either hot or cold. Gelatin media may be kept liquid at moderate temperature and ad- 
justed like liquid media. The addition of agar to a medium should not appreciably 
affect the Ph of the medium, so that an agar medium may be adjusted before the addition 
of the agar. Blood serum or whole blood, added to an alkaline or acid medium, tend to 
approximate the final Ph to 7.3 or 7.5. Such an addition of serum tends to hold the H- 
ion concentration stable during growth of bacteria, owing to the presence of the "buffer" 
salts. As Clark and Lubs have pointed out, the adjustment of culture media by the colori- 
metric method should be checked by the electrometric methods, where absolutely accur- 
ate results are desired. However, for the usual run of bacteriological work, the colorimetric 
method suffices. The usual adjustment of culture media is to a Ph of 7.4. While the 
optimum and limiting value for the reaction of culture media in the case of the various 
organisms have not been worked out. Fennel and Fisher have shown the following: Bacillus 
typhosus and B. paratyphosus grow on agar as alkaline as Ph 9.6 and on one of an acidity 
of Ph 4. The optimum range for these organisms is 6.2-7.2. The pneumococcus seem 
to have a range of 7.2 to 8.2. Streptococci grow well at 7.6 to 7.8. Meningococcus has a 
rather narrow range of good growth, from 7.4 to 7.8. Gonococcus has an optimum of 7.6 
The diphtheria bacillus, according to Davis, grows well in bouillon showing an hydrogen 
ion range of 6 to 8.1. See Clark and Lubs, Jour. Infect. Dis., 1915, XVII, 160; Jour. Biol. 



782 ' DIAGNOSTIC METHODS 

to +1 by adding the calculated amount of normal sodium hydrate. Boil two 
minutes over free flame, stirring constantly. Restore loss by evaporation and 
filter through paper or absorbent cotton. Titrate and accurately adjust the 
final reaction to +1. Place the broth in test-tubes, using 10 c.c. in each tube. 
Plug tubes with cotton and sterilize in the autoclave. 

Sugar-free Broth. 

Make the infusion of meat (do not use meat extract) as above. Add to 
this 10 to 20 c.c. of a 24-hour broth culture of bacillus coli communis and incu-' 
bate at 37°C. for 18 hours. Boil the mixture to kill the organisms and pro- 
ceed as above, using. this material as if it were ordinary meat infusion. 

Sugar Broths. 

Add to the sugar-free broth above mentioned i per cent, of dextrose, lac- 
tose or other sugar. Adjust the reaction of these broths so that it is neutral 
to phenolphthalein. Sterilize by the discontinuous method.^ 

Nutrient Gelatin. 

To the nutrient broth add 10 per cent, of ''gold label" sheet gelatin. 
Warm on water bath, stirring till gelatin is dissolved but not allowing the 
temperature to rise above 6o°C. Heat over steam bath for 30 minutes and 
restore loss by evaporation. Titrate, after boiling one minute to expel carbon 
dioxid, and adjust the reaction to -f-i as above described. Boil two minutes 
over free flame, stirring constantly. Make up loss by evaporation and filter 
through paper or absorbent cotton. If a clear filtrate be not obtained, it may 
be assured by cooling the mixture to 6o°C. and stirring in the white of an egg 
beaten up in 30 c.c. of water. Titrate and adjust final reaction. Tube as 
above and sterilize in the autoclave at i2o°C for five minutes. On being re- 
moved from the autoclave the gelatin is placed at once in the ice-box. 

Nutrient Agar. 2 

Prepare the meat infusion as given under nutrient broth. Weigh the fil- 
tered infusion and add 2 per cent, of Witte's peptone. Warm on water bath 
stirring until the peptone is dissolved and not allowing the temperature to go 
above 6o°C. While this mixture is being prepared, boil 15 grams of thread 
agar in 500 c.c. of water for one-half hour and make up the weight to 500 
grams. Let it cool to 6o°C. To 500 grams of the meat-peptone infusion 
add 500 grams of the 3 per cent, agar, keeping the temperature below 6o°C. 
Heat over steam bath for 30 minutes and restore loss by evaporation. 

Chem., 1916, XXV, 479; Jour. Bacteriol., 1917, II, i, 109, and 191; Lubs and Clark, Jour. 
Wash. Acad. Sc, 1915, V, 609; 1916, VI, 481; Hurwitz, Meyer, and Ostenberg, Bull. Johns 
Hopk. Hosp., 1916, XXVII, 16; Kligler, Jour. Bacteriol., 1917, II, 351; Barnett and 
Chapmann, Jour. A. M. A., 1918, LXX, 1062; Davis, Jour. Lab. and Clin. Med., 1918, 
III, 75; Kligler, Jour. Bacteriol., 1919, IV, 35; Avery and Cullen, Jour. Exper. Med., 1919, 
XXX, 359; Lord and Nye, Ibid., 389; Cohen and Clark, Jour. Bacteriol., 1919, IV, 407; 
Mcintosh and Smart, Lancet, 1919, II, 723; Jones, Jour. Infect. Dis., 1919, XXV, 262; 
Fennel and Fisher, Ibid., 444. 

1 See Holman, Jour. Infect. Dis., 1914, XV, 209; also Vedder, Ibid., 1915, XVI, 385.^ 

2 See Report of Committee on Standard Methods of Water Analysis, Jour. Infect. Dis., 
1907, Supplement III, 108; also, Huber, 111. Med. Jour., 1915, XXVIII, 207. 



CLINICAL BACTERIOLOGY 783 

Titrate and proceed as given above for nutrient gelatin. Great difficulty is 
sometimes experienced in filtering this preparation, but if the filter paper 
and funnel be wet with boiling water, the medium usually passes through 
easily. Sterihze in the autoclave. 

Glycerin Agar. 

Prepare nutrient agar in the usual way. After filtering add 6 to 8 per 
cent, of glycerin. Mix thoroughly, readjust the titer if necessary and place 
in test-tubes. Sterilize in autoclave. This medium is preferable to plain 
agar in cultivating such organisms as the tubercle bacillus, which does not 
grow at a-11 well on plain agar. 

Lactose and Dextrose Litmus Agar. 

To nutrient agar, prepared from sugar-free broth instead of nutrient broth, 
I per cent, of lactose or dextrose may be added just before sterilization, which 
must be in the Arnold sterilizer. Adjust the reaction to the neutral point to 
phenolphthalein. If the medium is to be used in tubes, the sterilized litmus 
solution or preferably azolitmin solution shall not be added until just before 
the final sterihzation. If it is to be used in Petri dishes, the sterilized solu- 
tions shall not be added until the medium is' ready to pour into the dishes. 
The litmus solution used is a i per cent, solution of Merck's purified litmus 
extract. Of this 5 or 6 per cent, is added. It is, probably, preferable to 
use instead of this, a i per cent, solution of Kahlbaum's azolitmin. 

"Ameba" Agar. 

This is the medium used by Musgrave and Clegg in cultivating the ameba 
and differs from nutrient agar in its strength. It is prepared in the same 
way but has the following formula: 

Extract of beef, 0.3 to 0.5 gram. 

Sodium chlorid, 0.3 to 0.5 gram. 

Agar, 20 . o grams. 

Water, 1,000.0 c.c. 

The reaction is made — i, that is, it is i per cent, alkaline. Variations may 
be made in this medium, where the very delicate organisms are concerned, by 
adding a very small amount of peptone. 

Blood Agar. 

Prepare about 40 tubes of nutrient agar and, while still unsolidified, place 
them in a slanting position so that a large surface is given. Such tubes are 
called slants. Place 50 c.c. of nutrient agar in a flask and melt it. Allow it 
to cool to about 45°C. and add 15 c.c. of human blood, which has been drawn 
by venous puncture. Mix the contents of the flask by shaking and pour 2 c.c. 
of this mixture over the surface of each agar slant. Place these slants in 
position again so that the added material may harden. These tubes cannot 
be resterilized. 

Instead of this method, which is preferable, one may add i to 2 c.c. of the 
human blood directly to 10 c.c. of agar melted and cooled to 40 or 45°C. 



J 



II 
i 



Hilt 



784 DIAGNOSTIC METHODS 

Mix by shaking and slant the tubes. This medium is especially valuable for 
routine work as it will grow practically all organisms. 

Ascitic or Hydrocele Agar. 

Dissolve 15 grams of agar in 1,000 c.c. of water. Filter and place 5 c.c. 
in each test-tube. Sterilize in the autoclave. To each agar tube add 5 c.c. 
of sterile ascitic or hydrocele fluid and mix by rotating the tube. Cool so as 
to form slants. This medium cannot be resterilized. It is not always 
the simplest procedure to prepare this medium as the body fluid may be 
contaminated. 

Litmus Milk. 

Fresh milk, as pure as possible, is obtained and steamed for fifteen min- 
utes in the Arnold. It is then placed in the ice box over night to allow the 
cream to separate. The cream is drawn off and the skimmed milk used for 
the medium. The reaction should be adjusted to +1. Add i per cent, 
azolitmin solution and tube 10 c.c. in test-tubes and sterilize for five minutes 
in the autoclave or in the Arnold for three successive days. 

Blood-serum. 

This is the medium devised by Loffler for the cultivation of the diphtheria 
organism and is very frequently used for others as well. It is prepared by 
adding i part of neutral nutrient broth containing i per cent, dextrose to 3 
parts of sterile beef-serum. The mixture is then mixed thoroughly and 
placed in culture boxes or, preferably, in short tubes. In the latter it should 
be slanted. Coagulate at 75°C. After being held at this temperature 
until coagulated, the serum may be very slowly heated to about 95°C. 

The above are the more important culture media used in routine work. 
Special media will be mentioned in discussing specific organisms. 



IV. Incubation 

Although bacteria may develop at temperatures above that of 37°C. 
(body temperature) or at much lower temperatures, yet the optimum tem- 
perature for most bacteria is 37°C. It is true that many organisms develop 
more luxuriantly and more rapidly at 2o°C., but these bacteria are, for the 
most part, non-pathogenic. 

It is essential, therefore, that some sort of apparatus be at hand, which 
will permit of the maintenance of a regular and uniform temperature of 37°C. 
Such an arrangement is called an incubator, many types of which may be ob- 
tained. Some of these are heated by gas and others by electricity, the latter 
being by far the more preferable. A heat-regulator is attached so that varia- 
tions in the temperature inside the incubator may be reduced to a minimum. 
In these incubators must be placed a dish of water to supply the necessary 
moisture, the development of bacteria proceeding the better the more nearly 
saturated the atmosphere of the incubator. 

Cultures when made upon the various media are placed in the incubator 



^ m 



CLINICAL BACTERIOLOGY 



785 



for periods ranging from 24 hours to 10 days or more depending on the organ- 
ism under investigation. As a rule 24 or 48 hours is the usual period of in- 
cubation. Owing to the rapidity of liquefaction of the medium, gelatin-con- 
taining media cannot be placed in a temperature of 37°C. Such a medium 
must be used at room temperature (about 2o°C.). 




Fig. 168. — Incubator. 



V. Preparation of Cultures 

As previously stated, oxygen is absolutely essential for the development of 
all bacteria. Some organisms are capable of growth in free oxygen (atmos- 
pheric air), while others cannot live in such an atmosphere but obtain their 
oxygen from the culture media upon or in which they develop. The former 
bacteria are known as aerobic organisms, while the latter types are called 
anaerobic bacteria. The majority of the pathogenic organisms belong in the 
aerobic division, although many important pathogens are anaerobic. 




786 DIAGNOSTIC METHODS 

A. Aerobic Organisms. 

In this group we find the diphtheria bacillus, tubercle bacillus, influenza 
bacillus, typhoid bacillus, cholera spirillum, gonococcus, meningococcus, 
pneumococcus, and many others. 

By means of the platinum needle, which is absolutely indispensable in 
bacteriological work, the material to be investigated is spread over the surface 
of the proper culture medium (in routine work agar or blood agar) and the 
cotton plug replaced. These plugs do not prevent access of air but keep out 
dust and contaminating bacteria. Place in the incubator and allow the 
culture to develop for 24 or 48 hours. Instead of the medium in the tubes, 
one may use liquefied agar or blood agar. Place the liquid material under 
investigation (such as milk or water) in a sterilized Petri dish and pour in the 
liquefied medium, which has cooled down to 40 or 45°C. 



Fig. 169. — Platinum Needles. 

Study the macroscopic characteristics of the culture and prepare smears 
from the culture for microscopic examination. If the cultures are pure, that 
is if only one type of organism is present, sub-cultures may be made upon 
special media in order absolutely to identify the organism. If several types 
are present, each kind of colony is transferred to other media and isolated 
in pure culture and then identified by sub-cultures. Where simple diagnosis 
is desired, these sub-cultures are rarely necessary, but where a pure culture is 
at issue, as in the preparation of a vaccine, very careful differentiation is 
essential. 

B. Anaerobic Organisms. 

In this class one finds the tetanus bacillus, treponema pallidum, Vincent's 
spirillum, organism of anterior poliomyelitis and many others. 

Cultures are made as in the case of aerobic organisms. However, these 
anaerobic types must develop in complete absence of free oxygen. To insure 
the absence of oxygen four methods are available. 

(i) Boiling the culture media. Allow the medium to cool down to 40 to 
45°C. and inoculate. This method is not at all reliable, owing to the possible 
access of air after boiling. 

(2) Displacement of the oxygen of the air by an inert gas, usually by hy- 
drogen. Inoculate the culture medium as above. Place in a larger vessel 
provided with entrance and exit tubes and tightly sealed. Pass a stream of 
purified hydrogen through the vessel continuously for several hours, close the 
stoppers and place the apparatus in the incubator. 

(3) Absorption of the oxygen. This is the more commonly employed 
method. Place the culture tubes on a glass or metal support inside a larger 



CLINICAL BACTERIOLOGY 



787 



tube or bottle. Place in the bottom of the bottle 10 grams of pyrogallol and 
pour on it 100 c.c. of i per cent, sodium hydrate solution. Tightly stopper 
the bottle and place in the incubator.^ 

(4) The use of a vacuum. This method consists in exhausting, with an 
air pump, the air in a large vessel containing the inoculated culture media. 
Close the tubes to prevent reentrance of air and place in the incubator. After 
the air is exhausted, hydrogen may be passed into the vessel, thus giving 
double assurance of freedom from oxygen. 




Fig. 170. — Anaerobic Cultivation in Hydrogen Gas (Heinemann). a, Novy jar; b, 
glass cock; c, gas generator; d, sulphuric acid; e, sodium hydrate solution; /, opening of 
Novy jar; g, stopper. 

After the cultures have developed they are studied and examined as men- 
tioned above. Sub-cultures and isolation of the different types are not so 
frequently essential with these anaerobic organisms, as the contamination is 
usually due to aerobic bacteria, which do not, of course, develop in these 
cultures. 

VI. Staining 

The general methods of staining bacteria depend upon the special organ- 
ism under investigation. As a rule Loffler's methylene blue (see p. 21) and 
Gram's stain (see p. 742) are the ones which give all the general informa- 
tion desired. Certain organisms, however, require special staining, which 
methods will be discussed under the specific organism. ^ Organisms are di- 
vided into two groups according to their reaction to Gram's stain, so that the 
writer would advise the constant use of this stain in order properly to classify 
an organism as positive or negative to this stain. 

Staining of Spores 

Certain organisms show within their protoplasm a small bright refractile 
spot, known as a spore. These are very resistant and develop into vegetative 
orgianisms very rapidly. The spore is not stained readily, but when once 
stained is very resistant to decolorization, the body of the bacillus losing its 

^ The following method is very successful for routine work. Push the cotton plug into 
the tube a short distance, place dr}^ pyrogallic acid on the upper portion of the cotton and 
pour on it 5 drops of 10 per cent, sodium hydrate solution. Stopper tightly with cork and 
seal it in with paraffin. 

2 See Fleischer, Jour. Med. Research, 1917, XXXVI, 31. 



ll« 



788 DIAGNOSTIC METHODS 

color before the spore does. In ordinary stained preparations the spore 
shows as an unstained spot in the body of the stained bacillus. Of course, the 
spore may be stained with the simple stain, if the action be prolonged or if 
the stain be heated, but this does not give clear differentiation.^ 

Moller's Method. 
Make a smear of the culture. Dry in air and fix in absolute alcohol for two 
minutes followed by chloroform for two minutes. Dry. Drop a few drops 
of 5 per cent, aqueous solution of chromic acid on the smear and leave for two 
minutes. Wash in water. Cover the smear with Ziehl's carbol fuchsin (p. 
20) and warm for one minute. Wash in water and decolorize in 5 per cent, 
sulphuric acid for a few seconds, completing the decolorization, if necessary, 
with absolute alcohol. Wash in water. Stain with methylene blue for one- 
half minute. Wash in water, dry and examine. The spores are stained red 
and the body of the organism is blue. 

Staining of Capsules. 

Many methods have been advocated for this purpose, but the writer has 
found the method of Rosenow (see p. 652) the most satisfactory, giving 
beautiful pictures and uniform results. 

Staining of Flagella. 
Van Ermengem's Method. 
f 11 I Place the film for five minutes at 5o°C. (or 30 minutes at room tempera- 

|l| 11 ture) in the following freshly prepared solution. 

( 20 per cent, aqueous solution of tannic acid, 60 c.c. 

fc 1 1 1 1 2 per cent, solution of osmic acid, 30 c.c. 

I im j Glacial acetic acid, 4 or 5 drops. 

\ Wash in water and then in absolute alcohol. Cover the smear with J'^ per 

cent, aqueous solution of silver nitrate for a few seconds and, without 
washing, transfer the slide to the reducing bath for a few seconds. This 
reducing bath is as follows: 

Gallic acid, 5 grams. 

Tannic acid, 3 grams. 

' , Fused sodium acetate, 10 grams. 

Jlllil Distilled water, 350 c.c. 

Without washing, place the slide in the }^ per cent, silver nitrate solution and 
move the liquid back and forth over the smear until this becomes black. 
Wash in water, dry and examine. 

Successful preparations by this method, or any other, depend upon the 
obtaining of thin specimens. 2 Take a small amount of a young agar culture of 
the organism and make a homogeneous emulsion in a watch glass with dis- 
tilled water. Place a drop or two of this emulsion on a perfectly clean glass 
slide and allow the drop to spread out. Dry in air and stain as above. 

1 See Huntoon, Jour. Am. Med. Assn., 1914, LXII, 1397; Jour. Immunol., 1916, I, 126. 

2 See Lancereaux, Presse Med., 1919, XXVII, 565. 



m 



CLINICAL BACTERIOLOGY 789 

VII. Identification of Organisms 

After cultures have been prepared as above described and have been incu- 
bated for 24 to 48 hours, the diagnosis of a given infection may be possible by 
direct examination of the cultures and stained slides made therefrom.^ This 
is true in cases in which search is being made for a specific organism without 
any reference to the associated bacteria. However, in the closer study of cul- 
tures, which is required in many cases, especially in the preparation of autoge- 
nous vaccines, sub-cultures on special media must be made in order properly 
to purify the culture and to permit of differentiation and recognition of the 
organism causing the infection. For this reason the writer deems it advisable 
to discuss briefly the cultural characteristics on special media and the means 
of absolute identification of some of the more important pathogenic bacteria. ^ 

I. Diphtheria Bacillus (Klebs-Loffler Bacillus). 

The medium best adapted for the development of the diphtheria bacillus 
is Loffler's blood serum. ^ By means of a stout platinum needle or a sterile 
swab remove a portion of the suspicious material or membrane from the nose, 
throat or tonsils and spread this over the surface of the above culture medium. 
If the material is to be sent to a laboratory and no culture medium be at hand 
place the swab in a sterile test-tube or homeopathic vial and plug this with 
cotton. It is very essential that the proper material be obtained for these 
cultures or otherwise negative results will obtain. If necessary tear away the 
membrane in order to be certain of your material. 

Place the culture in the incubator at 37°C. for six to eight hours and ex- 
amine. The cultures should rarely be examined after more than 20 hours to 
insure the best results. The growth appears as a mass of grayish-white 
points or colonies varying in size from a small to large pin-head. They are 
somewhat more opaque in their center than at their margin, which may be 
tinged a light yellow. The longer the period of growth, the larger the colonies, 
the more irregular their borders (early cultures usually show a regular margin) 
and the greater the tendency for the colonies to show radial striations. These 
colonies on blood serum always remain discrete and do not flow together. 

Smears made from such cultures are stained with methylene blue and with 
Neisser's or Gram's stain as given on page 39. Such smears show the organ- 
isms with the characteristic morphology and staining peculiarities given there. 
It is to be remembered that such cultures and smears may show no diphtheria 
bacilli, although the case is clinically one of diphtheria. This may be due to 

J"^ It is important to remember that transmutations may appear within the groups of bac- 
teria so that pneumococci, for instance, may change into true streptococci by variations in 
the culture medium, by changing the oxygen tension within the culture tube, etc. See 
Thiele and Embleton, Ztschr. f. Immunitatsforsch. und exper. Therap., 1913, XIX, 643; 
Rosenow, Jour. Infect. Dis., 1914, XIV, i; Jordan, Proc. Nat. Acad. Sc, 1915, I, 160. 

2 For a differential method of study of various bacteria by the addition of various 
aniline dyes to- the culture medium see Churchman, Jour. Exper. Med., 1912, XVI, 221; 
Ibid., 1913, XVII, 373; Churchman and Michael, Ibid., 191 2, XVI, 822; Krumwiede and 
Pratt, Ibid., 1914, XIX, 20 and 501; Simon and Wood, Am. Jour. Med. Sc, 1914, CXLVII, 
524- 

3 See Drigalski and Bierast (Deutsch. med. Wchnschr., 1913, XXXIX, 1237) who advise 
the, addition of ox bile to this medium. Davis, Jour. Lab. and Clin. Med., 1918, III, 
75; Davis and Ferry, Jour. Bacteriol., 1919, IV, 7\i\ Bunker, Ibid., 379. 



m 



fit 



790 DIAGNOSTIC METHODS 

the taking of improper material for the culture, to the fact that antiseptic solu- 
tions have been used prior to the time of taking the culture or to the fact that 
too long a period of incubation has been used. Under the latter condition 
other organisms will so far outgrow the diphtheria bacilli that the latter are 
unrecognizable. For these reasons it must be insisted upon that a negative 
bacteriologic examination should never be regarded as excluding diphtheria. 

In some very urgent cases recourse is made to the examination of direct 
smears from the swab without preliminary cultivation. This method rarely, 
if ever, gives results which may be relied upon but it occasionally does. It 
would seem to the writer that cultures should be insisted upon in all cases, as 
the time necessary for obtaining a growth is so slight that one is warranted in 
waiting for a more definite diagnosis. All suspicious throats, especially in 
children, should be regarded as diphtheritic until proven to be otherwise by 
several cultures. This precaution is best both for the patient and for his 
associates. See Schick's immunity test (p. 667). 

It not infrequently happens that certain organisms, such as Hofmann's 
bacillus (pseudo- diphtheria bacillus) and the xerosis bacillus, complicate the 
diagnosis, as these organisms resemble the diphtheria bacillus in many ways. 
Cultures of Hofmann's bacillus, grown onLoffier's medium, show smaller and 
whiter colonies than those of the diphtheria bacillus. The growth of the 
former is much more rapid and there is not the same tendency for the colonies 
to take up the pigment of the medium and become yellowish in color. If Hof- 
mann's bacillus be grown for 48 hours in glucose broth to which litmus has 
been added, no acid is produced; while the diphtheria bacillus readily pro- 
duces acid (red coloration of medium). This fact has been mentioned on 
page 40, whereon are given the different sugars upon which these conflicting 
organisms act with production of acid but no gas formation. This fermen- 
tative test is of great value in differentiation. 

Further, the morphology of Hofmann's bacillus is not the same as that of 
the Klebs-Loffler bacillus, although at times the two may become confusing. 
The former is shorter than the latter (although the latter occasionally shows a 
short form) ; the former is oval, stains deeply throughout except for one un- 
stained portion and shows no granules. Occasionally one may observe a few 
beaded forms but these are rare. The more or less regular morphology of the 
pseudo-diphtheria bacillus is quite in contrast to the pleomorphic type of the 
diphtheria bacillus. 

Negri and Mieremet^ and Bunting and Yates ^ report the successful cultiva- 

1 Centralbl. f. BakterioL, 1913, LXVIII, 292. 

2 Arch. Int. Med., 1913, XII, 236. See, also, Steele, Boston Med. and Surg. Jour., 1914, 
CLXX, 123; Billings and Rosenow, Jour. Am. Med. Assn., 1913, LXI, 2122; Kusunoki, 
Virchow's Arch., 1914, CCXV, 184; Yates, Bunting and Kristjanson, Jour. Am. Med. Assn., 
i9i4,LXIII, 2225; Verploegh, Kehrer and van Hoogenhuyze, Miinch. med. Wchnschr., 1914 
LXI, 1158; Mellon, Am. Jour. Med. Sc, 1915, CL, 245; Harris and Wade, Jour. Exper. 
Med., 1915, XXI, 493; Fox, Jour. Med. Research, 1915, XXXII, 309 and 325; Arch. Int. 
Med., 1915, XVI, 465; Rhea and Falconer, Ibid., 1915, XV, 438; Bloomfield, Ibid., 1915; 
XVI, 197; Bunting, Bull. Johns Hopkins Hosp., 1915, XXVI, 179; Olitsky, Jour. Am. Med. 
Assn., 191 5, LXIV, 1 134; Yates and Bunting, Ibid., 1953; Hatcher and Lemmon, Ibid., 
1915, LXV, 1359; Torrey, Jour. Med. Res., 1916, XXXIV, 65; Bunting and Yates, Bull. 
Johns Hopk. Hosp., 1917, XXVIII, 151; Cunningham, Am. Jour. Med. Sc, 1917, CLIII, 
406; Woolley, Jour. Lab. and Clin. Med., 1917, II, 523; Eberson, Jour. Infect. Dis., 1918, 
XXIII, I. 



CLINICAL BACTERIOLOGY 79I 

tion of a pleomorphic diphtheroid organism in cases of Hodgkin's disease, 
which organism had been previously demonstrated in the tissues by Frankel 
and Much. The former workers used Bordet's potato medium and call the 
organism isolated the Cory neb acterium granulomatis maligni, while the latter 
investigators employed Dorset's egg medium and glycerin-phosphate-agar 
and suggest the name of C or yneh acterium Hodgkini {Bacillus hodgkini). 
Rosenow has reported the growth of these organisms on Loffler's blood serum 
and blood agar. The morphology of this organism is very pleomorphic: 
thus plump short rods, some so short as to resemble cocco-bacilli; small thin 
bacilli with polar staining; comma-shaped bacilli; granular rods of variable 
size; branching forms; club-shaped involution forms and large spherical types. 
The organism is Gram-positive, is non-acid fast and does not form spores. 

2. Influenza Bacillus (Pfgiffer's Bacillus). 

This organism is found most frequently in the sputum and nasal dis- 
charges of those affected with influenza. It is often recognizable in the 
routine sputum examinations, but it is of ten overlooked owing to its small size. 

As hemoglobin is essential for the development of this organism, blood 
agar is the best medium upon which to cultivate it. ^ In obtaining the speci- 
men for culture work, select a solid portion of the sputum and wash it several 
times with sterile distilled water to remove the excess of mucus. Or, if de- 
sired, make an emulsion of the sputum in sterile water. Take up the solid 
part of the washed sputum and rub it over the surface of a blood-agar plate 
in a Petri dish. Incubate at 37°C. for 24 or 48 hours. Macroscopic ex- 
amination shows a number of small delicate dew-drop like colonies, which 
never become confluent. This organism is rarely found in pure cultures, 
the ancillary types being pneumococci, streptococci and staphylococci. ^ 
Other types of colonies will, therefore, be seen on the plates. Select a colony 
having the characteristics above mentioned and prepare smears as usual. 
The morphology and staining properties are as given on page 26. 

3. Pertussis Bacillus (Bordet and Gengou's Bacillus). 

The best medium for the cultivation of this organism is the potato medium 
of Bordet and Gengou, which is prepared as follows. To 200 c.c. of a 4 per 

1 Brown and Orcutt (Jour. Exper. Med., 1918, XXVIII, 659) advise streaking the plates 
with hemolytic streptococci as this seems to facilitate growth of the influenza bacillus. 
Avery (Jour. A. M. A., 1918, LXXI, 2050) has shown that a i :iooo sodium oleate added to 
the blood agar increased its value. This was prepared as follows: 5 c.c. of a 2 % solution of 
neutral sodium oleate were added to 95 c.c. of a meat infusion agar, i c.c. of a suspension 
of rabbit red blood cells was then added while agar was hot and plates were then poured. 
The optimum Ph for influenza bacilli is 7.2 to 7.5. See, also Pritchett and Stillman, Jour. 
Exper. Med., 1919, XXIX, 259; Winchell and Stillman, Ibid., XXX, 497. 

2 During the last few years quite an extensive epidemic of so-called "Spanish influenza" 
spread over the world, causing wide-spread destruction. The work on the epidemiology 
and etiology of this epidemic has been very extensive, the opinions being rather conclusive 
that the influenza bacillus was not entirely responsilbe for the trouble. I give only a few 
references, in this connection, as the mass of publications on the subject is too extensive to 
permit inclusion. See Mathers, Jour. A. M. A., 1916, LXVI, 30; Capps and Moody, 
Ibid., LXVII, 1349; Mathers, Ibid., 1917, LXVIII, 678; Bernstein and Loewe, Jour. In- 
fect. Dis., 1919, XXIV, 78; Duval and Harris, Ibid., XXV, 384; Hirsch and McKinney, 
Ibid., 394; Gay and Harris, Ibid., 414; Wollstein, Jour. Exper. Med., 1919, XXX, 555; 
Lucke, Wight and Krine, Arch. Int. Med., 1919, XXIV, 154; Duval and Harris, Jour. Im- 
munol., 1919, IV, 317; Parker, Ibid., 331; Valentine and Cooper, Ibid., 359. 



792 DIAGNOSTIC METHODS 

cent, aqueous solution of glycerin add loo grams of finely chopped potato. 
Heat in the autoclave for five to ten minutes. The supernatant fluid is now a 
concentrated glycerin extract of potato. To 50 c.c. of this extract add 150 c. c. 
of a 0.6 per cent, sodium chlorid solution and 5 grams of agar. Heat this 
mixture in the autoclave and, while hot, filter it into test-tubes, adding 2 to 3 
c.c. to each tube. Sterilize in the autoclave. Allow the medium to cool 
down to 40 to 45°C. and add an equal amount of sterile defibrinated human 

1^ . „ or rabbit blood. Mix thoroughly and prepare slants of the mixture. This 

11 iB medium is especially valuable, also, in cultivating the influenza bacillus, 

gonococcus and meningococcus. 

This bacillus should be looked for in the early stages of the disease when 
coughing begins. In the later stages is is not so easily found owing to the 
large number of other bacteria. Select a solid piece of the sputum and wash 
in sterile distilled water. Spread it over the surface of a Petri plate prepared 
from the above medium. These plates are more satisfactory for the bacilli 
of pertussis and influenza than are the slants, as these organisms require 
much oxygen. The primary cultures are difficult to obtain, as the growth 
is so scanty during the first 24 hours, and often in 48 hours, that little is 
visible. Sub-cultures made from this initial growth give a more copious 
development. On the third day the colonies appear white, slightly elevated 
and sharply outlined. From generation to generation the growth becomes 
quicker and more luxuriant. The morphology and staining characteristics 
of this organism have been given on page 26. 

So close is the similarity of the influenza and the pertussis bacilli that it 
seems advisable to differentiate these organisms.^ The growth of the former 
is quicker and less luxuriant than that of the latter. The colonies of influenza 
bacilli are slightly bluish and transparent, while those of the pertussis 
organism are whiter and thicker. If stroke cultures are made upon slanted 
potato-blood-agar, the growth of the pertussis bacillus is thicker than that of 
-' the influenza bacillus; its border is abrupt while that of the influenza bacillus 
is more spread out. The growth of the influenza bacillus is always trans- 
parent, shows an irregular border and has a moist appearance. Further, the 
pertussis bacillus hemolyzes the blood medium, so that the line of culture ap- 
pears clearer than the surrounding. The influenza bacillus does not cause 
hemolysis. 

Maflory, Horner and Henderson (Jour. Med. Research, 1913, XXVII, 
391) have shown the etiologic relationship of this organism to pertussis, 
finding it in the characteristic lesions between the cilia of the epithelial 
cells lining the trachea and bronchi. 

4. Typhoid Bacillus (Eberth-GafEky Bacillus). 

The characteristic cultural properties of this organism have been discussed 

on pages 330 and 648, while its agglutination reactions are given on page 669. 

The isolation and detection of the typhoid bacillus in the various excretions 

and secretions of the body is a matter of considerable difficulty, often calling 

^ See Morse, Boston Med. and Surg. Jour., 191 6, CLXXV, 723; Chievitz and Meyer, 
Ann. de I'lnst. Pasteur, 1916, XXX, 503. 



CLINICAL BACTERIOLOGY 793 

upon all the resources at the command of the worker. Study of the cultural 
characteristics, of the agglutination reactions and the staining properties of 
this organism will readily differentiate it from the colon and associated 
bacteria, but careful work is necessary. It is questionable whether the ty- 
phoid bacillus has been isolated from water and milk supplies in a pure state, 
but this is not at all necessary as the detection of the colon bacillus, which 
is more easily identified, is all that is necessary to establish pollution. The 
typhoid bacillus may be found in the dejecta of convalescent and recovered 
patients for some time, so that careful measures should be adopted to prevent 
spread of disease through these carriers.^ 

5. Colon Bacillus (Bacillus Coli Communis). 

This organism has been discussed on page 650 and its cultural character- 
istics and points of differentiation given. The colon bacillus is coming into 
great prominence, owing to its discovery in so many of the secretions and ex- 
cretions of the body under pathological conditions. ^ The use of vaccines 
made from cultures of colon bacilli is varied and the results usually excellent. 

In the bacteriological examination of milk and water, the detection of the 
colon bacillus is of especial importance as an indicator of sewage contamina- 
tion. In times past the custom has been to use, for this work, litmus-lactose 
agar for the plate cultures and glucose broth for the study of the fermentative 
properties of the organisms in the water. It is wxll known that both water 
and milk, as well as other fluids, contain acid- and gas-producing organisms 
which are not colon bacilli, so that one is not justified in calling all such 
bacteria colon bacilli. In order to eliminate these extraneous gas- and acid- 
producing bacteria, the use of bile has been advocated. The addition of bile 
to the usual media inhibits the other organisms but was not supposed to 
affect the colon bacilli. The media used in this later study and now adopted 
for all sanitary examinations of water and milk supplies are the lactose bile 
medium, consisting of i per cent, lactose dissolved in ox bile and sterilized, 
and lactose-bile- agar. The bile agar is prepared as follows. In 1,000 c.c. 
of fresh neutral ox bile dissolve 15 grams of agar, 10 grams of Witte's peptone 
and 10 grams of lactose, boiling as little as possible. When dissolved, filter 
the medium without titration, place in tubes and sterilize in the autoclave for 
three minutes. Rector^ has advocated a slight modification of this medium, 
which he claims is much better. His formula is as follows: 

Dried ox bile, 100 grams 

Witte's peptone, 10 grams 

Agar, 15 grams 

Lactose, 10 grams 

Neutral red (i per cent, solution), 10 c.c. 

Distilled water, 1000 c.c. 

1 See Jordan, Jour. Am. Med. Assn., i9i4,LXII, 1772; Hirschbruch, Berl.klin.Wchnschr., 
1914, LI, 1176; Garbat, Jour. A. M. A., i9i6,LXVII, 1493; Gumming, Ibid., 1917, LXVIII, 
1163. 

2 See Gonradi and Bierast, Kolle and Wassermann's Handbuch, 1913, VI, 483; also 
Bornand, Gentralbl. f. Bakteriol., x\bt. 2, 1913, XXXVIII, 516. 

^ Am. Jour. Pub. Health, 1913, III, 154. 



794 DIAGNOSTIC METHODS 

While it is unquestionably true that this bile medium has an advantage 
over non-bile media in that interfering organisms are eliminated and, there- 
fore, the acid- and gas-producing organisms may more certainly be classified 
as colon bacilli, yet Jordan^ has shown that this medium exerts an inhibiting 
effect on colon bacilli themselves to the extent of 333^^ to 50 per cent, of the 
viable colon bacilli present in the material under investigation. Such being 
the case much more care must be exercised in reporting upon a specimen of 
water or milk, especially as to numbers of colon bacilli and as to the safety of 
such specimens for general use. 

In recent years a vast amount of work has been done on the differentia- 
tion of the members of the colon group. From this work it appears that cer- 
tain characteristics, such as the gas ratio, the Voges-Proskauer reaction and 
the Methyl Red Test of Clark and Lubs are especially valuable in this differ- 
entiation. Rogers, Clark and Davis and Rogers, Clark and Evans, using a 
slight modification of the accurate method of Keyes for the collection and 
examination of the gases produced by members of this group in carbohydrate 
media, show that bacteria of the colon-aerogenes family may be sharply 
divided into two groups on the basis of the gas ratio. One group produces 
CO2 and H in the constant ratio C02:H2 = 1.06, the members of this group 
being termed ''the low ratio organisms." The second group produces much 
more CO2 than H and furnishes a ratio which varies from about 1.90 to 3.00, 
such organisms being called "the high ratio organisms." In this study there 
were also found some organisms, which produce CO2 but no hydrogen. 
These organisms may be distinguished from the colon-aerogenes family by 
cultural characteristics. As they liberate no hydrogen, the gas ratio is 
infinity, the organisms being termed "the infinity ratio group." 

The Voges-Proskauer reaction is a colorimetric test, observed after addi- 
tion of 10 per cent, sodium hydrate to the fluid in the fermentation tube for 
the purpose of absorbing the CO2 and thus, determining the gas ratio. If 
the alkalinized carbohydrate medium, in which organisms of this group have 
been grown, be allowed to stand for 24 hours at room temperature a reddish 
color resembling eosin will appear, when this Voges-Proskauer reaction is 
positive. ' While it is customary to allow the tube to stand for 24, and by some 
for 48 hours, yet this would seem to be unnecessary as Levine, Weldin, and 
Johnson have shown that a faint reaction may be recognized within i hour 
and, after 5 hours, as distinct a reaction as at the expiration of 24 hours. 
The true colon bacilli give negative reactions with this test, while the bacillus 
cloacae and associated members of this group show a positive result. That is 
a positive Voges-Proskauer reaction is given by those members of the "high 
gas ratio group," showing large production of CO2 and small amounts of H. 
This reaction appears to be due to a special decomposition of glucose with 
the formation of acetyl-methyl-carbinol (CH3-CHOH-C.O-CH3). This 
product is further oxidized, in presence of air, to diacetyl ((CH3CO)2), which 
in the presence of NaOH reacts with the peptone of the culture medium to 
form the eosin-like compound. Levine, Weldin, and Johnson have^ intro- 

^ Jour. Infect. Dis., 1913, XII, 326. 



CLINICAL BACTERIOLOGY 795 

duced a fuchsin-aldehyd reaction, which is, also, used in the correlation of 
members of this group. The test is performed as follows: To 2 c.c. of a 72 
hour culture of the organism in the 0.5 per cent, carbohydrate-pep tone-dipo- 
tassium phosphate medium of Clark and Lubs were added 2 or 3 drops of basic 
fuchsin decolorized with sodium sulphite. A slight pink coloration is recorded 
as negative, while a distinct red or cherry color is considered positive. As 
compared with the results of the Voges-Proskauer reaction, this latter test 
shows that among 124 cultures, which gave the positive Voges-Proskauer, 
only 10 were positive with the fuchsin test; while 44 or 45 cultures of Voges- 
Proskauer negative strains showed positive results with the fuchsin test. 

Clark and Lubs have introduced a further colorimetric test, which serves 
to differentiate members of the colon-aerogenes family. The medium used 
in this test consists of 0.5 per cent. Witte peptone, 0.5 per cent, pure dextrose, 
and 0.5 per cent. K2HPO4. While the authors state that Witte peptone 
must be used, other workers have found other peptones quite as effective, 
especially the peptone of Parke, Davis & Co. and the Difco product. The 
test is performed as follows: Pour about 5 c.c. of the culture in the 10 c.c. of 
the above culture medium (which has been incubated for at least 3 days) into. 
a clear glass test tube and add 2 to 4 drops of paranitrophenol solution (0.2^ 
gram dissolved in 30 c.c. alcohol and diluted to 500 c.c. with distilled water) 
and add i to 2 drops of methyl-red solution (o.i gram dissolved in 300 c.c, 
alcohol and diluted to 500 c.c. with distilled water) to the remaining 3^-^ of the 
culture medium. Record the color changes. In a study of 190 cultures hy 
this method, the following results were noted: Without exception, the "low 
ratio ©rganisms "(true colon bacilli) were perfectly colorless to paranitrophe- 
nol and brilliantly red to methyl red; without exception the "high ratio organ- 
isms" were distinctly colored with paranitrophenol, while all but five were 
distinctly yellow to methyl red. In other words the "low ratio cultures" 
are distinctly acid, while the "high ratio cultures" are distinctly alkaline. It 
is to be said that the Methyl Red gives sharper results, so that this is recom- 
mended over the paranitrophenol. In comparing this test with the Voges- 
Proskauer, we find that positive methyl red organisms show negative Voges- 
Proskauer reactions and vice versa. The test is really a method of distin- 
guishing the H ion concentration of the culture media in which members of 
this group are grown. ^ 

6. Dysentery Bacillus (Shiga-Kruse Bacillus). 

In bacillary dysentery certain organisms are found which closely resemble 
the typhoid and colon bacilli in morphology but which differ in certain cul- 

^ In this connection see Voges and Proskauer, Ztschr. f. Hyg., 1898, XXVIII, 20; 
Harris, Bull, de I'lnst. Pasteur, 1906, IV, 250; Harden, Proc. Royal Soc, 1906, LXXXVII, 
424; Keyes, Jour. Med. Res., 1909, XXI, 69; Frieber, Centralbl. f. BakterioL, Abt.i, 
Orig., 1913, LXIX, 437; Kligler, Jour. Infect. Dis., 1914, XV, 187; Browne, Ibid., 580;. 
Rogers, Clark and Davis, Ibid., 1914, XIV, 411; Rogers, Clark, and Evans, 1914, XV,. 
99; Ibid., 1915, XVII, 137; Clark and Lubs, Ibid., 160; Levine, Ibid., 1916, XIX, 773;; 
Levine, Weldin, and Johnson, Ibid., 1917, XXI, 39; Burton and Rettger, Ibid., 162; 
Nelson, Jour. Am. Chem. Soc, 1917, XXXIX, 515; Bronfenbrenner and Davis, Jour. 
Med. Res., 1918, XXXIX, 33; Koser, Jour. Infect. Dis., 1918, XXIII, 377; Salter, Ibid.^ 
1919, XXIV, 260; Winslow, Kligler, and Rothberg, Jour. Bacteriol., 1919, IV, 429. 



796 



DIAGNOSTIC METHODS 



tural characteristics from the latter bacteria. In the course of an epidemic of 
dysentery in Japan, Shiga discovered this bacillus, but it remained for Kruse 
to elaborate and fix its characteristics. Since that time, similar organisms 
have been found in various epidemics throughout the world, but in many 
cases certain differences were observed in the behavior of the organisms to- 
ward different sugar-containing media. On the basis of fermentative tests 
with acid production in sugar-broths, and, also, upon variability in the agglu- 
tination reactions of the organisms isolated, at least four distinct types of the 
dysentery bacillus must be considered as established. 

I. Shiga-Kruse type. 
II. Flexner-Strong type. 

III. Hiss-Russell, El Tor I strain, "Y" type. 

IV. Harris- WoUstein type. 

The following table, taken from Jordan,^ will show at a glance the varia- 
tions in the action of these similar organisms. 



Bacillus dysenterise 




Acid production from: 


Indol 


Mannite 


Maltose 


Saccharose 


Dextrose 


duction 


Type I (Shiga-Kruse) . . 






+ 


+ 
+ 

+ 




Type II (Flexner-Strong) 

Type III (Hiss-Russell) 


+ 


- 


-f 
-1- 


Type IV (Harris-WoUstein) 


+ 


+ 


+ 


+ 



The detection of these organisms in the stool is not a matter of great 
difficulty, by cultural methods, if the case is examined relatively early in its 
progress. However, after some time the examination becomes more and 
more uncertain until the organisms are rarely found after the third week. 
The material for the preparation of cultures is obtained from a freshly passed 
stool. Flecks of mucoid material are selected and placed in sterile water 
or physiologic salt solution, in order to remove the colon bacilli with which 
the material is contaminated. This washing is repeated three or four times, 
fresh water being, of course, added each time. If there be no visible mucus 
in the stool, emulsify a portion of the stool in nutrient broth. The washed 
mucoid particles, or a portion of the fecal emulsion, are now rubbed over the 
surface of three or four litmus-lactose agar plates or of plates made of Drigal- 
ski and Conradi's medium (see p. 131) without the addition of the crystal 
violet. Allow these plates to dry in the air by keeping the cover slightly 
open for one-half hour, place them in the incubator at 37°C. with the bottom 
side up and examine at the end of 24 hours. Any colon bacilli left after the 

^ General Bacteriology, Philadelphia, 1913. See, also, Lentz, Kolle and Wassermann's 
Handb., 1913, III, 927; Barber, Philippine Jour. Sc, 1913, VIII (B), 539; Bluhdorn, Deutsch. 
med. Wchnschr., 1914, XL, 782; Monatsschr. f. Kinderhkde., 1914, XIII, Orig., 37; Schild, 
Ibid., 51; Musgrave and Sison, Philippine Jour. Sc, 1914, IX (B), 241; Flexner and Amoss, 
Jour. Exper. Med., 1915, XXI, 515; Sutherland, Brit. Med. Jour., 1916, II, 142; Diinner 
and Lauber, Berl. klin. Wchnschr., 1916, LIII, 1266; Foucar, Mil. Surg., 1916, XXXVIII, 
534; Smillie, Am. Jour. Dis. Child., 191 7, XIII, 337; Gibson, Jour. Royal Army Med. 
Corps, 1917, XXVIII, 615. 



CLINICAL BACTERIOLOGY 797 

washing, and there are always some, will appear as red colonies with a gradual 
shading of color into the blue medium. The Shiga bacillus does not change 
the color of the medium, its colonies appearing as translucent, iridescent, 
dew-drop like growths with irregular margins and centers more opaque 
than the edges. This grow^th is very similar to that of the typhoid bacillus 
on this medium. Test one of the latter colonies for motility of the organisms 
by the hanging drop method and, also, test its agglutination with a potent 
dysentery serum. If these tests are satisfactory, namely, if no motility is 
observed and agglutination is definite, sub-cultures of these colonies are made 
upon slant agar, neutral red-glucose-agar (glucose agar to which three or 
four drops of a saturated aqueous solution of neutral red are added), litmus- 
milk and plain milk. The cultures on agar and neutral red-glucose-agar 
show the same characteristics as do those of the typhoid bacillus, but the 
lack of motility excludes this latter organism. These cultures have a peculiar 
sperm-like odor. On litmus-mannite-agar the Shiga bacillus produces no 
acid, while the typhoid bacillus develops a distinct red color. Litmus-milk 
is not changed in color, nor is plain milk coagulated. 

It wall be seen, therefore, that the Shiga bacillus differs from the typhoid 
bacillus in its action toward litmus-mannite-agar and, especially, in its lack of 
motility and its different agglutination reactions. From the colon bacillus, 
the cultural characteristics differentiate it sharply. 

The morphology and staining properties of this organism have been given 
on page 132. It is Gram-negative, does not form spores, nor does it possess 
flagella. In smear preparations, the dysentery bacillus resembles the typhoid 
bacillus, but the former is thick and often polymorphous. 

Culturally the other types of dysentery bacilli are differentiated from th6 
Shiga bacillus by the fact that their cultures on litmus-lactose-agar are more 
irregular and have a reddish- violet tinge, while the latter organism shows colo- 
nies of a translucent dew-drop like appearance. Further, the Shiga bacillus 
does not produce indol, while all the other types, as well as the colon bacillus, 
do. In confirming this differential diagnosis, the agglutination tests are 
valuable. The Shiga bacillus should agglutinate in a dilution of i to 20 of the 
patient's serum, while the Flexner type agglutinates at a dilution of i to 80. 
For differentiation of the other types, the fermentative tests must be utilized, 
as agglutination is not typical. 

7. Bacillus Aerogenes Capsulatus. 

This organism, which is also known as the Bacillus Welchii and the ^^Gas Bacil- 
lus,''^ has assumed considerable importance during the recent war owing to the 
number of cases of infection arising from contamination of wounds with it. 
Hitherto it has been known as a contaminating organism of soils and drinking 
water and has been especially associated with cases of emphysematous gan- 
grene. During the war, the infection with this organism reached such a degree 
owing to the fact that the clothing of the men was contaminated w^ith the 
organism or its spores due to the trench life and, also, to the fact that the soil 
had been so intensely cultivated prior to the war. An interesting report on 



798 DIAGNOSTIC METHODS 

the contamination of the clothing has been made by Simonds^ who states 
"Spores of the Bacillus welchii group of bacteria were found on loo per cent, 
of the uniforms of Belgian soldiers who had come directly from the trenches, 
and in the meshes of all the samples examined of the new cloth from which 
the uniforms were made." In a study of infection with this organism Taylor'^ 
suggests that the fatal cases of infection pass through the following phases: 
Phase I, dormant stage; localized infection of the wound; Phase 2, stage of 
acute gaseous distention, the result of obstruction to the escape of the gas 
generated locally in the wound; Phase 3, explosive stage; this is characterized 
by the rapid extension of the swelling associated with subcutaneous crepita- 
tion: Phase 4, stage of systemic intoxication; Phase 5, stage of septicemia. 
The terminal invasion of the blood by the baciUi, when it happens, is probably 
a very late phenomenon, occurring approximately at the time of death. 
The first stage represents the common type seen in fresh wounds. The great 
majority of cases do not pass beyond this stage. When, however, for any 
reason, the free escape of the gas becomes interfered with, the second phase, 
with its clinical picture of gas gangrene develops rapidly and passes frequently 
within a few hours into the third stage. If sufficient drainage be established 
at this point for the escape of gas, and too large a mass of muscle has not been 
destroyed, recovery is usual. It is at this time or before that the value of X- 
ray examination is so great from the diagnostic point of view. It is true that, 
while the opportunities for infection with this organism were so great, there 
was a relatively small incidence of gas gangrene in infected wounds. The 
reasons for this were, probably, the rapid surgical interference which was 
given and the drainage of the wound. Bulloch and Cramer^ would have us 
believe that certain influences, particularly chemical, produced local changes 
leading to local breakdown of the normal defensive mechanism against the 
bacteria of this group. For this phenomenon, they have coined the word 
" cataphvlaxis " or "defense rupture." 

The gas bacillus is widely distributed, being commonly found in the intesti- 
nal tract of the higher animals, and in soil, dust, sewage, river-water and milk. 
While the cause of emphysematous gangrene, it has been observed in cer- 
tain uterine infections, and infections of the gastro-intestinal, genito-urinary, 
and biliary tracts (Jordan). The development of gas in the internal organs, 
especially the liver, the so-called "foamy organs," observed post-mortem is 
often due to this organism. As Jordan* states, great confusion has reigned 
in the field of identification and nomenclature. The following names prob- 
ably refer to the same organism: B. welchii, B.aerogenes capsulatus, B.phleg- 
monis emphysematosae, B. enteritidis sporogenes, and B. perfringens. A 
bacillus found by Achalme and others in cases of acute rheumatism, and re- 
•garded by them as standing in causal relation to that affection, is almost cer- 
tainly identical with B. welchii." 

^ Jour. Exper. Med., 1917, XXV, 819. 
2 Jour. Path, and Bacteriol., 191 7, XX, 384. 
^ Proc. Royal Soc. London, 1919, XC, 513. 
^ General Bacteriology, Saunders, Phila. 



CLINICAL BACTERIOLOGY 799 

This organism was originally described by Welch and Nuttall^ and later 
by Fraenkel. It is a plump, rather long bacillus (3 to 6/j.), occurring in chains 
and singly; it is non-motile, anaerobic, and stains by Gram's method. Cap- 
sules are usually present in preparations made from the organs or body fluids. 
Spores are formed by some races and are particularly prone to appear on 
blood-serum. Gas is produced in dextrose, lactose, and saccharose media, 
but not in mannite; a small amount of gas may be formed, also, from protein 
substances. Hydrogen predominates in the gas produced from sugar media, 
the ratio of H and CO2 ranging from 2:1 to 3:1. Milk is coagulated with 
abundant gas production and strongly acid reaction (stormy fermentation); 
the casein is not digested. The acids produced are butyric and allied acids, 
but lactic acid is scanty. For this reason, there is a typical odor of butyric 
acid in milk and glucose-agar cultures. Most varieties liberate hemoglobin 
when grown in bouillon to which blood has been added. Smith has advocated 
the simple method of detecting this organism by adding a bit of sterile normal 
liver or other animal tissue to broth in the fermentation tube. Gas usually 
develops abundantly in 24 hours at 37°C., if the inoculating material con- 
tains the spores of B. welchii. This organism grows well on all ordinary 
culture media, rather rapidly at 37°C., but is strictly anaerobic. The colon- 
ies are greyish-white on agar, and gelatin is liquefied. In gluocse-bouillon 
the growth is first evidenced by a diffuse cloudiness with the fluid later 
becoming clear and a whitish viscid sediment settling out. While there are 
some similarities between this organism, especially as observed in water and 
milk analyses, and the bacillus coli communis, yet there should be little 
difficulty in differentiating them by recourse to the above cultural methods 
and a search for the spores of the B. welchii. 

While this organism is pathogenic for man, under certain conditions, yet 
it does not affect the lower animals universally. Rabbits and mice are 
practically immune to simple inoculation. Dead rabbits are, however, of 
special value in a study of this organism. If a suspected material be injected 
intravenously into a rabbit and the rabbit be killed a few minutes thereafter 
and the animal incubated at 37°C., gas is produced in a few hours throughout 
the body, the liver showing gas within 4 to 6 hours after inoculation. Guinea 
pigs show a variable reaction; if injected subcutaneously they sometimes die 
with subcutaneous emphysema accompanied by extensive necrosis and tissue 
digestion; sometimes they develop local abscesses; and sometimes are 
unaffected. Pigeons are, also, highly susceptible. 

8. Cholera Vibrio (Koch's Comma Bacillus). 

This organism has been briefly discussed on page 129. It appears in 
stained preparations as a small, short slightly curved rod, which has led to the 
name of '' comma" bacillus. It is about 2 ju long and 0.5 ^t broad. It is not 

^Bull. Johns Hopk. Hosp., 1892, III, 81; Fraenkel, Centralbl. f. Bakt., 1893, XIII, 13' 
see, also, Welch and Flexner, Jour. Exper. Med., 1896, I, 5; Dunham, Bull. Johns Hopk- 
Hosp., 1897, VIII, 68; Welch, Ibid., 1900, XI, 185; Kamen, Centr. f. Bakt., 1904, XXXV, 
554 and 686; Arch. f. Hyg., 1905, LIII, 128; Herter, Jour. Biol. Chem., 1906, II, i; 
McCampbell, Jour. Infect. Dis., 1909, VI, 537; MacNeal, Latzer, and Kerr, Ibid., 571; 
Blake and Lahey, Jour. A. M. A., 1910, LIV, 167 1. 



8oo DIAGNOSTIC METHODS 

infrequent to observe long, straight involution forms, especially from liquefied 
gelatin cultures. This bacillus has a single flagellum at one end and shows 
very active motility. It stains easily with the ordinary stains and is Gram- 
negative. 

The spirillum of cholera grows well on practically all media at room tem- 
perature, with the exception of potato.^ The medium must be alkaline in re- 
action to insure the growth. This alkalinity is obtained by adding 3 c.c. of 
10 per cent, sodium hydrate to 100 c.c. of neutral medium. 

The growth on gelatin plates at room temperature is more or less char- 
acteristic. These colonies appear, after 24 hours, as round and regular white 
points, which later become irregular and show a granular appearance as if the 
colony were covered with ground glass. Within 48 hours liquefaction begins, 
the colony sinking into the depression. On gelatin stab-cultures, the lique- 
faction begins as a cup-shaped depression at the surface, a bubble of air being 
retained in the depression. The later liquefaction takes the form of a funnel, 
being much broader at the surface. Later the medium is entirely liquefied. 
Some of the vibrios resembling the cholera bacillus, as especially the Finkler- 
Prior vibrio, produce a more rapid liquefaction of the gelatin and do not show 
the bubble of air which is practically always present in the cholera cultures. 
This point is not, however, distinctive enough for strict diagnostic purposes. 
It is to be remembered, in this connection, that the other types of organisms 
found in the intestinal canal do not liquefy gelatin media. 

On agar plates the colonies appear as pale, flat, thin growths which are 
transparent and show, by transmitted light, a peculiar opalescence. As the 
cholera spirillum is a strongly aerobic organism, we find a copious surface 
growth in i per cent, peptone solution containing 3^^ per cent, sodium chlorid. 
Other organisms do not develop readily in this medium, so that we not in- 
frequently may obtain an almost pure culture by use of this simple medium. 
The addition of a few drops of concentrated sulphuric acid to such a culture 
produces a burgundy-red color, due to the formation of nitroso-indol. Thus 
the cholera spirillum produces indol and, at the same time, reduces the nitrates 
of the culture medium to nitrites. While the colon bacillus produces indol, it is 
necessary to add a trace of nitrite before the nitroso-indol reaction is obtained. 
This cholera-red reaction, as it is called, was supposed to be distinctive for the 
cholera vibrio but it has been shown that other cholera-like organisms also 
react. A negative result with this test is, therefore, of greater value than a 
positive one. 

In the bacteriological diagnosis of cholera, several points must be borne in 
mind. Select some of the '' rice particles " and inoculate a peptone water cul- 
ture as above, or plant i c.c. of fecal material in 50 c.c. of this medium. From 
this surface growth, at the end of six to eight hours at 37°C., sub-cultures are 
made upon other media and smears are examined and studied for the char- 
acteristic organisms. Glycerin and agar plates are inoculated and incubated 
for 24 to 48 hours, the former at room temperature and the latter in the incu- 
bator. It should be a rule to inoculate Dieudonne's alkali-hlood-agar kt the 

' See Teague and Travis, Jour. Infect. Dis., 1916, XVIII, 601. 



CLINICAL BACTERIOLOGY 8oi 

same time. This medium is prepared by mixing equal parts of defibrinated 
beef blood and normal (4 per cent.) sodium hydrate; heat in autoclave for 
one-half to three-fourths hour. Add 30 parts of this mixture, while still hot, 
to 70 parts of neutral 3 per cent. agar. Mix and sterilize in the autoclave. 
When cooled, pour into Petri plates. Allow the plates to dry by standing 
open in incubator for several hours and do not use these plates earlier than, 
24 hours after preparing. Colon bacilli and other extraneous organisms do 
not grow on this medium, while the cholera organism develops rapidly. The 
colonies are large, circular, crystal-clear by transmitted light and by reflected 
light gray. Microscopic examination of the organisms from these colonies 
shows many degenerative forms, very rarely any typical comma forms. 

After these cultures are prepared test the motility of the organism in hang- 
ing drop and follow this by the agglutinating reactions and, especially, by the 
application oi Pfeiffer's bacteriolytic test. This latter test is as follows: An 
emulsion of the organisms in question is injected into the peritoneal cavity 
of a guinea-pig, which has been immunized against a definite species of the 
vibrio. In 20 to 30 minutes remove, with a trocar, some of the peritoneal 
fluid and examine under the microscope. The organisms present will be non- 
motile and appear as more or less granular spherical masses instead of the 
typical forms, if the reaction is positive; if negative, the organisms will appear 
as in the usualy preparations.^ 

9. Gram-positive Cocci. 
A. Pneumococcus (Diplococcus of Fraenkel; Streptococcus Pneumoniae). 

This organism is practically always found in the normal throat, so that its 
presence therein does not indicate a pathologic process. It is found, also, as 
the primary etiologic factor, in a wide range of infectious processes, so that it 
must not be regarded as limited to pneumonic types of infection. The mor- 
phology and staining characteristics of this organism have been given on page 
25, while its cultural peculiarities, especially, have been discussed on page 651. 
It is to be remembered that, from the morphological standpoint, it is often- 
times impossible to distinguish the pneumococcus from the streptococcus, as 
the former not infrequently appears in chains, and the typical lanceolate 
diplococci give place to forms which are more nearly true coccoid types. 

Cultural methods must, therefore, be employed and the special peculi- 
arities of the pneumococcus and streptococcus differentiated by the use of the 
blood-agar plates, the blood bouillon tubes, the use of bile acids, and the 
fermentative test with inulin as outlined on page 652. The agglutination 
test should, also, be employed with specific immune serum in all cases of 
doubtful diagnosis. 2 

It is to be remembered that there are distinct varieties of pneumococci, 
which are distinguishable from one another only by careful study of the cul- 

^ See "The Bacteriological Diagnosis of Cholera," Pub. Health Rep., 1Q12, XXVII, 371; 
also, Kolle and Schiirmann, Kolle and Wassermann's Handb., 1912, IV, i; Kabeshima, 
Centralbl.f. Bakteriol., Abt. i, i9i3,LXX, 202; Craster, Jour. Exper. Med., 1914, XIX, 581. 

2 See Dochez and Gillespie, Jour. iVm. Med. Assn., 1913, LXI, 727; also, Floyd and 
Wolbach, Jour, Med. Research, 1913, XXIX, 493; Dochez and x\very. Jour. Exper. Med., 
1915, XXI, 114; Lyall, Ibid., 146. 



8o2 DIAGNOSTIC METHODS 

tural peculiarities as well as by observation of their effects on animals. Tran- 
sition forms from the pneumococcus to the streptococcus and vice versa are 
observed under certain cultural conditions, so that it is often a more than diflGi- 
cult problem properly to classify these organisms. One of these organisms, 
the streptococcus mucosus capsulatus, is probably to be considered with the 
true pneumococci. It occurs as round, or very slightly lance-shaped cocci, 
which are encapsulated, the capsule being wider than that of the pneumo- 
coccus. The organisms usually appear in pairs, but there are always seen a 
few in chains of four to eight organisms. The colonies on blood agar have a 
mucoid slimy consistency and have a greater tendency to become confluent 
than do those of the true pneumococcus. 

. B. Streptococci. 

There are several varieties of streptococci, which are not readily distin- 
guished by their morphology but which cultural peculiarities usually suffice to 
identify. These cultural points have been discussed on pages 653 and 654. 
It is to be said, however, that this differentiation is not a simple proposition, 
as these varieties readily change, the one into the other and, also, into forms 
which are hardly distinguishable from the typical pneumococcus. The chief 
types of the streptococcus are 

a. Streptococcus pyogenes (hemolyticus) , a long-chained form which pro- 
duces no green coloration on blood agar but shows a large colorless hemolytic 
area about its colonies. 

b. Streptococcus viridans, a short-chain type producing green colonies on 
blood agar but only slight hemolysis. 

c. Streptococcus mucosus, an encapsulated organism discussed above, pro- 
ducing no green colonies nor any hemolysis. 

The standard routine method,^ proposed by a special committee, for the 
isolation and identification of hemolytic streptococci from throats, sputa, 
and pathologic exudates, is very precise and clear-cut. This method involves 
the isolation of the hemolytic streptococcus into bouillon from a blood agar 
plate and the use of the bouillon culture for the following tests: (i) A study 
of the staining characteristics of the organism and the length, size, shape 
and arrangement of the cocci in the chains; (2) study of the ability of the 
organisms to lake the corpuscles of a rabbit. 0.5 c.c. of the bouillon culture 
is mixed with 0.5 c.c. of a 5 per cent, suspension of washed rabbit corpuscles 
in physiologic salt solution and incubated in a water-bath at 37°C. for 2 hours. 
Streptococci which lake the blood completely in this time are to be regarded 
with suspicion; (3) to about i c.c. of bouillon culture add 3-^ volume of ster- 
ile ox bile. Observe for i hour at room or incubator temperature. Solubil- 
ity in bile, serves to distinguish certain strains of pneumococcus from the 
streptococcus, which does not dissolve in the bile; (4) fermentation reactions 
toward lactose, mannite, salicin, inulin, raffinose, and saccharose should be 
determined; (5) a study of the power of the organism to coagulate milk should 
be made, incubation being continued for 7 days; finally, (6) a study of the 

' Holman, Avery, Kinsella, and Brown, Jour. Lab. and Clin. Med., 1918, III, 618. 



CLINICAL BACTERIOLOGY • 803 

effects of inoculation of rabbits or mice should be made, the rabbits being 
inoculated intravenously and the mice intraabdominally. 

Many other types have been isolated in pathological conditions and their 
exact classification is rather difficult.^ Usually the organisms have shown 
characteristics which place them in classes intermediate between those given 
above and even have indicated their close relationship to the pneumococcus. 
One of these virulent organisms has been discussed on page 41, being called 
the streptococcus epidemicus and causing streptococcic sore-throat. 

In these later days much work has been done on this group of organisms 
and much of value has been forthcoming. Some of this work is so important 
that the writer must discuss it briefly, in order properly to correlate the sub- 
ject of streptococci to various pathological processes. 

Davis^ has called attention to the close association of streptococcic infec- 
tions to chronic arthritic troubles and finds that the atrium of infection in 
these arthritides is usually the tonsil or peritonsillar structures. As is dem- 
onstrated th« streptococcus hemolyticus, the diplococcus (streptococcus) 
rheumaticus and the streptococcus epidemicus (see p. 41) are very prone to 
attack the joints; while the streptococcus viridans rarely does so. Such cases 
can be separated by the clinical findings and history of tonsillitis in the pa- 
tients from the other types of deforming arthritides. The bacteriologic ex- 
amination of cultures from the tonsil clears up the etiology. In taking the 
cultures from the tonsil, the material must be obtained from the depths of 
the crypts after the tonsil has been incised. In such cultures the streptococ- 
cus hemolyticus is usually found. The streptococcus viridans is found in 
surface cultures, but this is disregarded as it is less virulent and is rarely 
associated with arthritic involvements. It is to be recalled that the strepto- 
coccus hemolyticus may be found on the surface of the tonsils in ordinary 
streptococcic tonsillitis, and even on normal tonsils, but these do not neces- 
sarily have any special general value in the absence of clinical history of joint 
troubles. In the treatment of these cases of arthritis due to streptococcic 
infection, proper vaccines often work like magic. Two hundred million of 
the killed organisms are given at a dose with five-day intervals between 
the administrations. 

Rosenow^ in his work on the pneumococcus and streptococcus has isolated 

HJlrich, Jour.-Lancet, 1915, XXXV, 627; Rosenow, Jour. A. M. A., 1915, LXV, 1687; 
Ibid., 1916, LXVII, 662; Detweiler and Robinson, Ibid., 1653; Blake, Jour. Exper. Med., 
1916, XXIV, 315; Holman, Jour. Med. Res., 1916, XXXIV, 377; Becker, Jour. Infect. 
Dis., 1916, XIX, 754; Krumwiede and Valentine, Ibid., 760; Smillie, Ibid., 191 7, XX, 
45; Blake, Jour. Med. Res., 1917, XXXVI, 99; Holman, Am. Jour. Med. Sc, 1917, CLIII, 
427; Kinsella and Swift, Jour. Exper. Med., 191 7, XXV, 877; Ibid., 1918, XXVIII, 169 
and 181; Pilot and Davis, Jour. Infect. Dis., 1919, XXIV, 386; Nakayama, Ibid., 489; 
Bryan, Ann. Otol. Rhinol. and Laryngol., 1919, XXVIII, 337; Nichols, Ibid., 344; Avery, 
Dochez and Lancefield, Ibid., 350; Russell, Ibid., 374; Tongs, Jour. A. M. A., i9i9,LXXIII, 
1277. 

2 Jour. Infect. Dis., 1912, X, 148; Jour. Am. Med. Assn., 1913, LXI, 724. See, also, 
Richards, Ibid., 1914, LXII, no; Holman, Jour. Path, and Bacteriol., 1915, XIX, 478. 

3 Jour. Am. Med. Assn., 1913, LX, 1233; Jour. Infect. Dis., 1914, XIV, i and 61; 
Rosenow, New York Med. Jour., 1914, XCIX, 270; Jour. Am. Med. Assn., 1914, LXII, 
1146; Ibid., 1914, LXIII, 903; Davis, 111. Med. Jour., 1914, XXVI, 158; Billings, Ibid., 164; 
Jour. Am. Med. Assn., 19 14, LXIII, 899; Smith and Brown, Jour. Med. Research, 1914, 
XXXI, 455; Hopkins and Lang, Jour. Infect. Dis., 1914, XV, 63; Thro, Ib"'d., 34; Holman, 



8o4 DIAGNOSTIC METHODS 

certain organisms from infected joints, blood cultures and tonsillar infections 
in cases of acute articular rheumatism and finds organisms corresponding 
to the micrococcus rheumaticus of Poynton and Payne. He finds, however, 
that this organism resembles very closely the streptococci, its virulence being 
less than that of the streptococcus hemolyticus and greater than that of the 
streptococcus viridans. By animal passage and other means three of the 
strains isolated from acute rheumatism have been converted into typical 
hemolytic streptococci on the one hand and into pneumococci on the other. 
The cultures made from the involved joints and regional lymph nodes have 
revealed three types of streptococci. One produces green colonies on blood 
agar and forms long chains; a second produces a narrow zone of hemolysis 
and forms short chains; while the third forms a grayish-brown colony with- 
out affecting perceptibly the blood in the medium. This latter organism 
appears as a diplococcus, in short chains and as single cocci. The most 
distinctive cultural feature of these types is their production of a very high 
acid reaction in dextrose-broth and abundant growth at low temperatures. 
Progressive transmutation from a streptococcic type to a pneumococcus is 
easily shown. 

Billings,^ on the basis of this work of Davis and Rosenow, has studied the 
clinical aspects of arthritic cases and finds that a chronic focal infection is as- 
sociated with many of these arthritidfes. Clinically, he finds the focal dis- 
ease in the faucial tonsil, dental alveoli or jaw, antra of head, etc., in a large 
number of cases. In other cases chronic colon, gonorrheal or streptococcic 
infection of the seminal vesicles and prostate were found to be the focal point 
of the arthritic involvement. Cultures were prepared from the material 
obtained from the focal infection, sub-cultures made and the dominant organ- 
ism isolated in pure culture. From these cultures vaccines were prepared 
and used with excellent results. Billings is especially emphatic upon the 
point of using the auto-vaccine from the dominant isolated organism and 
believes that only by this method may good result from vaccine treatment. 

C. Staphylococcus Pyogenes. 

These organisms grow well on all types of media. Their cultures on agar 
are best adapted to the development of the pigment, which is the character- 
istic differentiating point of members of this species. Cultivation at room 
temperature frequently brings out the pigment better than growth in the 
incubator. These organisms are readily recognized, in stained preparations, 
by their morphology. They appear as distinct cocci, five to fifty in a cluster 
often compared to a bunch of grapes. ^ Rarely do they occur singly or in pairs. 
Occasionally short chains may be observed, but most of them are clumped. 
In purulent material these organisms may be both intra- and extracellular, 
but their staining characteristics readily distinguish them from the Gram- 
negative types of organism. 

Ibid., 293; Crabtree, Ibid., 309; Davis, Ibid., 378; Rosenow, Ibid., 1915, XVI, 367; Thro., 
Ibid., 1915, XVII, 227; Broadhurst, Ibid., 277; Brawley, 111. Med. Jour., 1915, XXVIII, 
178. 

1 Arch. Int. Med., 191 2, IX, 484; Jour. Am. Med. Assn., 1913, LXI, 819. 

2 See Walker and Adkinson, Jour. Med. Res., 191 7, XXXV, 373. 



CLINICAL BACTERIOLOGY 805 

10. Gram-negative Cocci. 
A. Gonococcus (Diplococcus of Neisser). 

The morphological and staining characteristics of the gonococcus have 
been discussed on page 741. It is to be emphasized that this organism can- 
not, in every case, be distinguished by its morphology, staining characteristics 
and intracellular position from other organisms, especially from the meningo- 
coccus, micrococcus catarrhalis and a few saprophytic diplococci, such as 
diplococci flavus, crassus and cinereus.^ In judging of the possibility of a 
certain organism being the gonococcus, the origin of the culture must be 
known and something of the clinical history must be given. Further, it is 
to be recalled that the intracellular grouping of the organisms is not especially 
limited to the gonococcus nor is it always present in every gonorrheal case. 
Such arrangement is given both by the meningococcus and micrococcus 
catarrhalis. In the early cases of gonorrhea, when pus cells are very few in 
number, the gonococci are for the most part extracellular, some of them lying 
upon the epithelial cells, but practically none being found within the pus cells. 
As the pus cells increase in number, the intracellular types become predomi- 
nant owing to the active phagocytosis. In the later stages and in the chronic 
forms of infection, the extracellular types become more frequent, very few 
intracellular forms being observed. 

As the gonococcus does not grow readily, if at all, on the ordinary culture 
media, special media is necessary. Albumin, preferably of human origin, i& 
essential in any medium to be used. The best medium is Wertheim's serum 
agar prepared by adding i part of human serum to 2 to 3 parts of nutrient 
agar. The serum is warmed to 4o°C. and added to the liquefied agar of the 
same temperature. Instead of the serum, ascitic or hydrocele fluid may be 
substituted as suggested by Kiefer and Steinschneider. In fact, ascitic agar 
is probably more frequently used than is the serum agar. Blood agar also 
affords an excellent medium for the development of the gonococcus. 2 

On the above media, colonies of the gonococcus appear in 24 hours as gray- 
ish-white points, which merge later into growths of a grayish-blue tinge with 
slight iridescence. These colonies have a slimy consistency, are round but 
soon become confluent. The gonococcus is very sensitive to drying, so that 
the culture medium must have plenty of water of condensation. It survives 
exposure to air for only a short time, although in masses of dried pus it may 
live, exceptionally, for six or seven weeks. In favorable culture media it 
rarely maintains its vitality more than 48 to 72 hours at room temperature 
but will live longer if kept in a refrigerator (Jordan). 

The gonococcus is bacteriologically identified by its characteristic morphol- 
ogy, its Gram-negative staining, its frequent intracellular position and by its 
ready growth on blood or ascitic agar and its lack of growth on ordinary media. 

1 See Kligler, Jour. Exper. Med., 1913, XVII, 653. 

2 For a discussion of the Human plasma glucose-agar medium of Thomson, the Tryp- 
tamine-blood-extract agar and broth of Cole and the Trypsinized pea-extract agar of Gor- 
don and Hine see Special Report No. 19, Medical Research Committee of National Health. 
Insurance, London, 1918. See also, Wherry and Oliver, Jour. Infect. Dis., 1916, XIX, 
288; Watabiki; Ibid., 733; Thomson, Brit, Med. Jour., 191 7, I, 869. 



8o6 DIAGNOSTIC METHODS 

These characteristics are, however, not sufficient to differentiate it from the 
closely related organisms, which will be discussed below. It is to be said that 
a diagnosis of a gonorrheal infection outside of the genital tract is never safe if 
based entirely on microscopic evidence, and the cultural evidence is not always 
clear. The micrococcus catarrhalis is not infrequently the etiologic factor 
in a genito-urinary infection as well as in other parts of the system, while the 
meningococcus very often causes certain genito-urinary complications, which 
may be associated with the presence of this latter organism in this tract (see 
Koch^). Further, in chronic cases of true gonorrhea, it is very frequently a 
difficult matter to detect the gonococcus either microscopically or culturally. 
The organisms found may be Gram-variable and few will be intracellular, so 
that great care must be exercised in diagnosis. In such cases one should use 
the provocative test of administering some irritant or allowing the patient 
to partake freely of alcohol. Such a procedure followed by milking the pros- 
tate will usually result in the appearance of typical gonococci in the purulent 
material expressed. Of course in these cases one may use the complement- 
fixation test or the vaccination test as suggested by Irons, and later, by Fron- 
stein (see p. 667). 

From the clinical point it may tax the resources of the physician to estab- 
lish a diagnosis of gonorrhea in any given case, outside of those cases of acute 
gonorrhea in which the history and symptomatology are clear. However, in 
cases of meningitis with genito-urinary complications or of general systemic 
gonorrheal infection with meningeal symptoms, the diagnosis is not so simple, 
as a differentiation must be established between the meningococcus and 
gonococcus. 

In this differentiation, cultivate the material obtained from the urethral 
discharge or from lumbar puncture upon faintly alkaline ascitic agar for 24 to 
48 hours. The meningococcus grows more luxuriantly than the gonococcus. 
The colonies of the former are larger (2 to 3 mm. in diameter) than those of 
the latter (i to 1.5 mm.). On microscopic examination of these colonies with 
the low power, the colonies of the meningococcus are found to be flat, homo- 
geneous, usually round, very slightly irregular border but never notched; 
while those of the gonococcus are irregular, border usually notched and center 
always elevated. On cultivating these organisms on litmus-ascitic-agar 
media containing various sugars, the gonococcus will show acid production in 
the dextrose media only; while the meningococcus produces acid in both dex- 
trose and maltose, but in no others. 

The micrococcus catarrhalis, with which the gonococcus may be often con- 
fused, grows well on ordinary media. It does not produce acid in any sugar- 
containing medium. Its morphology, staining characteristics and intra- 
cellular position are, however, exactly like those of the gonococcus. 
B. Meningococcus (Diplococcus Intracellularis Meningitidis). 

The morphological and staining characteristics of this organism of 
Weichselbaum have been given on page 759- For the bacteriological diagno- 
sis of a meningococcic infection, one may use spinal fluid, blood, or purulent 

^ KoUe and Wassermann's Handbuch, 1913, IV, 68q. 



CLINICAL BACTERIOLOGY 807 

products from metastatic points, secretions of nose and bronchi. This ma- 
terial should be examined as soon as possible after obtaining, as the meningo- 
cocci are not especially resistant to various changes. 

For the development of this organism, especially of the first generation, 
media are necessary which contain animal, preferably human, albumin. The 
blood agar or ascitic agar given above are the best to use. The cultural 
peculiarities of the meningococcus on this medium have been given. ^ From 
the cultures the organisms not infrequently appear variable in size and show 
variable depth of staining, although they are always Gram-negative. 

In examining spinal fluid for these organisms, centrifuge the fluid in a 
sterile tube and plant two or three ascitic agar plates with the sediment. Iso- 
late the colonies by sub-culture if necessary and identify the organism mi- 
croscopically. Inoculate special sugar-containing media and differentiate by 
the results mentioned above. The meningococcus produces acid only in 
dextrose and maltose media. 

In examining material from the nose or throat simple microscopical exami- 
nation is of little avail, as we have the possibility of finding the micrococcus 
catarrhalis as well as the saprophytic diplococci previously mentioned. Cul- 
tural work and differentiation on sugar media are absolutely essential. These 
saprophytic cocci, especially the diplococcus crassus, are found in both the 
lumbar fluid and nasal secretions. This latter diplococcus produces acid in 
dextrose, maltose, galactose, levulose, cane-sugar and lactose media. This 
organism is probably identical with Jager's meningococcus. Some of the 
varieties of the diplococcus flavus ferment levulose in addition to dextrose and 
maltose, while other types (Diplococcus flavus III) acts exactly like the men- 
ingococcus. ^ It will be seen, therefore, that the fermentation test should be 
applied in all differential work, where the meningococcus is concerned. 

C. Micrococcus Catarrhalis. 

This organism is discussed on page 17. It is found in catarrhal condi- 
tions of nose and throat and, also, in infections of the genito-urinary tract. 
It grows readily on all media. On ascitic agar plates the colonies are white, 
firm, irregular, dry and somewhat smaller than the growths of meningococcus. 
Under low microscopic power they appear brown and granular, while those of 
the meningococcus are yellow and homogeneous and of the gonococcus gray- 
ish and slimy. The firm and dry quality of the colony is characteristic of the 
micrococcus catarrhalis. From the ordinary staphylococci, it is differen- 
tiated by its non-liquefaction of gelatin. 

Preparations made from culture media show involution forms, some of 

1 See Cohen and Markle, Jour. A. M. A., 1916, LXVII, 1302; Douglas, Med. Jour. 
Australia, 1916, II, 382; Lloyd Jour. Path, and Bacteriol., 1916, XXI, 113; Gordon, Hine 
and Flack, Brit. Med. Jour., 1916, II, 678 and 682; Davison, Davison and Miller, Jour. 
Exper. Med., 191 7, XXVI, 779; Bushnell,' Jour. Med. Res., 191 8, XXXVIII, i. Binger 
(Jour. Infect. Dis., 1919, XXV, 277) has shown that methylene blue inhibits the growth of 
meningococci in fairly high dilutions. 

2 See Kutscher, Kolle and Wassermann's Handbuch, 1913, IV, 589. The meningo- 
coccus has been grouped, on the basis of immunological reactions, into 4 groups. See 
Ellis, Brit. Med. Jour., 1915, II, 881; Arkwright, Ibid., 885; Olmstead, Du Bois, Neal 
and Schweitzer, Jour. Immunol., 1916, I, 307; TuUoch,- Jour. Royal Army Med. Corps., 
1917, XXIX, 66; Vines, Jour. Path, and Bacteriol., 1918, XXII, 56. 



8o8 DIAGNOSTIC METHODS 

the organisms being very large. In the direct smears of purulent material 
the organisms are Gram-negative, intracellular or extracellular biscuit-shaped 
diplococci. For its identification cultural work is necessary and a study of its 
fermentative powers is essential. The micrococcus catarrhalis does not pro- 
duce acid in any sugar-containing medium. An organism described as the 
Micrococcus cinereus is probably identical with this organism of catarrh. 

VIII. Vaccines 

In previous sections of this book the problems of immunity in its relation 
to opsonins, agglutinins and precipitins have been discussed briefly. It was 
shown that the injection of a vaccine (a suspension, in physiologic salt solu- 
tion, of a certain number of killed organisms) into the body of a patient suffer- 
ing with a chronic infection due to the organism injected brought about a 
systemic reaction by which the resistance of the patient was increased. In 
this way it was hoped that the natural resources of the body would be brought 
into activity by the stimulation of antibody formation and that this would 
result in great benefit to the patient. Further, by the use of vaccines, the 
resistance of the human organism against bacterial invasion could be so in- 
creased that immunity was conferred, for a variable period of time, toward the 
organism so injected. The most recent use of this latter principle is the 
antityphoid vaccination, which will be discussed later. 

It will be remembered that patients, who have passed through an attack of 
an infectious disease, have acquired, in most infectious diseases, an immunity 
against reinfection. An attempt is often made, especially in the treatment 
of infections with diphtheria organisms, tetanus bacilli and meningococci, to 
aid the patient in his efforts to overcome the invasion, that is to help him in 
acquiring immunity, by injection of serum from an animal previously immun- 
ized against such infection. This serum or antitoxin supplies antibodies to 
the infected patient, with which he may overcome the toxic products of 
bacterial development in the system. In other words, serum therapy confers 
immunity, shown in recovery from the acute infection, by aiding the system 
through introduction of antibodies produced outside the system of the pa- 
tient. Vaccine therapy, on the other hand, produces immunity by causing 
the production of antibodies within the system itself. It must be recalled that 
patients who have been inoculated with serum (protein) often become sensi- 
tized to its action to such an extent that a later injection of the same serum, 
even in much smaller dosage, may provoke serious and even fatal results. 
This is the phenomenon of anaphylaxis and must be remembered whenever 
one is dealing with the parenteral introduction of protein material into the 
system.^ 

^ See Besredka, in Kraus and Levaditi's Handbuch, igii, Erganzungsbd. I, 209; Biedl 
and Kraus, Ibid., 255; Abderhalden, Ztschr. f. physiol. Chem., 1912, LXXXII, 109; Wells 
and Osborne, Jour. Infect. Dis., 1913, XII, 341 ; Weil, Jour. Med. Research, 1913, XXVIII, 
243; Auer and Van Slyke, Jour. Exper. Med., 1913, XVIII, 210; Zinsser, Ibid., 219; Vaug- 
han, Jour. Am. Med. Assn., 1913, LXI, 1761; Friedberger, Nederl. Tijdschr. v. Geneesk., 
1913, II, 1065 and 1278; Nixon, Brit. Med. Jour., 1913, II, 1351; Weil, Jour. Med. Re- 
search, 1913, XXIX, 233; Leschke, Ztschr. f. exper. Path. u. Therap., 1914, XV, 23, 
Seligmann, " Anaphylaxie," in Handb. d. Biochem. des Menschen u. d. Tiere, Erganzbd., 



CLINICAL BACTERIOLOGY 809 

In this section the writer wishes to discuss the method of preparation of 
vaccines, both of the stock and autogenous varieties. The elaboration of the 
principles upon which their use is based as well as a discussion of the thera- 
peutic value finds no place here. 

I. Preparation of Vaccines. 
A. Stock Vaccines. 

The material for this work is obtained, under the strictest precautions to 
prevent contamination, from a focus of infection with the organism whose 
vaccine is desired It is at once plated upon general or special medium, de- 
pending upon the organism in question, and the organism isolated in pure 
culture. This may be a simple matter or it may be a very complicated one. 
In any case, all the differential points previously given for the varying or- 
ganisms must be kept in mind, so that proper medium and methods of isola- 
tion may be employed. It is absolutely essential that the final culture be 
pure, if the vaccine is to be of any value. 

In order to obtain a large amount of the pure culture, transfers from the 
original pure culture are made to three or four tubes of the medium best suited 
for its development and these tubes incubated for 24 to 48 hours. The cul- 
tures are then washed from the surface of the medium, using sterile physio- 
logic salt solution as the menstruum and scraping the surface lightly with the 
platinum needle to insure the removal of the organisms. Pour this sus- 
pension into a sterile flask or large tube and shake thoroughly to break up the 
bacterial masses. This is an important point in the technic, as otherwise the 
suspension will not be uniform. If necessary, one may filter out the large 
clumps by means of a sterile filtering arrangement. 

Having prepared the suspension, this is then sterilized by heating in a 
water-bath to 6o°C. for one hour. From this tentatively sterilized suspen- 
sion, cultures are made upon proper media by transferring several loopsful of 
the suspension to the surface of the slanted media and incubating for 48 hours. 
If no growth occurs, the suspension may be regarded as sterile.^ 

Standardization. — The sterilized vaccine is now examined for the number 
of bacteria per c.c. of the suspension. The method usually adopted is to com- 
pare the number of bacteria in a definite volume of suspension with the num- 
ber of red blood cells in the same volume of freshly drawn blood. The technic 
is as follows. By means of a graduated capillary pipet place equal volumes of 

1913, 248; Vaughan, "Anaphylaxis and Infection," Harvey Lectures, 1913-14, p. 132; 
Bradley and Sansum, Jour. Biol. Chem., 1914, XVIII, 497; Wallace, Jour. Am. Med. Assn , 

1914, LXII, 1166; Longscope, Jour. Exper. Med., 1915, XXII, 793; Soula. Presse Med., 
1 91 6, XXIV, 471; Weil, Jour. Immunol., 1916, I, i, 19, 35 and 47; Klinkert, Nederl. 
Tijdschr. V. Geneesk., 1917, I, 202; Novy, DeKruif and Novy, Jour. Infect. Dis., 1917, 
XX, 499; Novy and DeKruif, Ibid., 536, 566, 589, 618, and 629; Jour. A. M. A., 1917, 
LXVIII, 1524; Zinsser and Parker, Jour. Exper. Med., 191 7, XXVI, 411. Newer work 
along the line of Vaughan's Protein Poison includes the following: Underhill and Hendrix, 
Jour. Biol. Chem., 191 5, XXII, 465; Pryer, Jour. Lab. and Clin. Med., 1916, 1, 490; Gum- 
ming and Chambers, Ibid., 428; Vaughan, Ibid., 643 and 851; Ibid., 1916, II, 15; Jour. 
A. M. A., 1916, LXVII, 1559. 

^ King and Davis (Am. Jour. Pub. Health, 1914, IV, 917) have shown that potassium 
tellurite is an excellent indicator of sterility. Characteristic black compounds are pro- 
duced if a little of the salt be added to fluids containing viable bacteria. 



8 10 DIAGNOSTIC METHODS 

blood and of bacterial suspension on a glass slide, mix thoroughly and spread 
as for an ordinary blood slide. Stain with Wright's or other blood stain. 
Count the number of red cells and of bacteria in a number of microscopic 
fields and obtain an average of each. As the number of red cells per cu. mm. 
is arbitrarily fixed at 5 million, the relation of bacteria to the red cells, as 
shown by the counts, will readily give the number of bacteria in the cu. mm. 
The number in i c.c. is obviously 1,000 times greater.^ 

Having obtained the concentration of the original suspension, the vaccine 
must be standardized so that each c.c. shall contain a standard number of 
bacteria. This is done by diluting with sterile physiologic salt solution 
(which contains sufiicient cresol to bring the concentration of the final product 
up to 0.2 to 0.4 per cent, cresol) until the proper strength is reached. The 
addition of the cresol prevents future contamination of the vaccine and is an 
additional safeguard as to sterility of the product. The standard maximum 
dosage per c.c. of some of the more usual stock vaccines is as follows: 
Staphylococcus, 500 million - 

Streptococcus, 100 million 

Colon bacillus, 100 million 

Gonococcus, 100 million 

Typhoid, 1,000 million 

Pneumococcus, 100 million 

Of course this maximum is not suited to all cases. The dosage must be varied 
for the individual and for the type of infection. 

B. Mixed Stock Vaccines. 

A prerequisite to the use of any vaccine should be the bacteriological 
identification of the infection in question. The use of a simple or mixed stock 
vaccine is never justified without such a precaution. It is questionable 
whether one should ever use a mixed stock vaccine, but if the bacteriological 
examination has revealed a mixed infection, a proper mixture may be used in 
the proportions suitable for each separate constituent of the vaccine. In 
recent years the number and varieties of mixed stock vaccines have increased 
to such an extent that they are to be regarded as questionable therapeutic 
agents. To quote from Billings, "There is a value in vaccine therapy but 
to obtain benefit from vaccines, they should be rationally used. To use 
vaccines, simple or mixed, in the treatment of a patient, without first ascer- 
taining the nature of the disease, and, if infectious, the kind of invading or- 
ganism, is unscientific, reprehensible and wrong. When the mixed vaccines 
or the filtrate of culture-broth of countless organisms are used, it is like the 
shotgun prescription of our ancestors of the profession. They were and are 
intended to hit something." Such usage must be regarded, therefore, as 
mere guesswork. 

1 See Hopkins (Jour. Am. Med. Assn., 1913, LX, 1615) for a method of standardizing 
vaccines based on centrifugation and determination of percentage of bacterial sediment; 
Glynn, Powell, Rees and Cox, Jour. Path, and Bacteriol., 1914, XVIII, 379; Blaker, Indian 
Jour. Med. Research, 1914, I, 726; Harvey and Acton, Ibid., 1914, II, 648; Fitch, Jour. 
Am. Med. Assoc, 1915, LXIV, 893. Dreyer and Gardner, Biochem. Jour., 1916, X, 399; 
Gates, Jour. Exper. Med., 1920, XXXI, 105. 



CLINICAL BACTERIOLOGY 8ll 

C. Autogenous Vaccines. 

These are the proper agents to use whenever a vaccine is to be employed. 
The specific toxins of the organism call into activity the specific antibodies, 
which are necessary to overcome the effects of the organism causing the 
infection, for which the autogenous vaccine is being prepared. "Instead of 
adding a heavier burden to the individual's immunizing mechanism, already 
taxed in its struggle against the invading bacteria, these bacteria, reintro- 
duced as autogenous vaccines, if successful stimulate further therapeutic 
immunizing responses." It can be considered as established that these 
autogenous vaccines are the ones giving the best results. 

These autovaccines are prepared from the discharges, excretions, blood, 
etc., of the patient. The effort to obtain pure cultures and definitely to 
identify the organism or organisms which are dominant must be painstaking, 
both aerobic and anaerobic cultures being made in many cases. If there be 
a mixed infection, each organism is isolated in pure culture and the suspen- 
sions of the pure cultures are then mixed in such a way that the resulting 
combination will contain the organisms in their usual strength per c.c. In 
this standardization of the autogenous vaccines a little more latitude is al- 
lowed than in the case of stock vaccines, as the dosage of the autovaccine 
must be adjusted more to the individual than to the standard. In other 
words, one must use these autovaccines in graduated dosage, beginning with 
a small dosage and working up. 

It is true that occasionally mixed autovaccines are prepared which have 
not been sufficiently differentiated. This method is uncertain and crude. 
The method employed is to obtain a culture of all the organisms present in the 
discharge and prepare a suspension which shall have a standardized content 
within the range of usefulness of the organisms present. Here we are con- 
fronted with the same proposition as in the case of mixed stock vaccines. 
All the organisms should be identified, if not they should not be used. In 
this way a more reliable and more scientific product will be furnished.^ 

2. Antityphoid Vaccination. 

The basis of the use of vaccines, as a preventive immunizing inoculation 
against typhoid fever, is the observation that one attack usually confers im- 
munity from further attacks. This use of vaccines has become widespread 
and is promising great results, especially in those subjected to variations in 
the sanitary surroundings. The doses are given at ten-day intervals; the 
first dose contains 500 million organisms and the second and third doses i 
billion bacteria each. A local reaction is usually present but this subsides 
within 48 hours, while a general systemic reaction is not always observed. 

^ See Klenk, Interstate Med. Jour., 1915, XXII, 1209; Warden, Jour. A. M. A., 1915, 
LXV, 2080; Babcock, Lancet-Clinic, 1916, CXV, 139; Hekman, Nederl. Tijdschr. v. Gen- 
eesk., 1916, I, 2157; Hess, Am. Jour. Dis. Child., 1916, XII, 466; Wohl, Am. Jour. Med. 
Sc, 1916, CLII, 262; Hektoen, Jour. A. M. A., 1916, LXVI, 1591; Fox, Ibid., 2064; Sewall 
Mitchell, and Powell, Ibid., 1916, LXVII, 95; Satterlee, Ibid., 1729; Davis, Ibid., 1917, 
LXVIII, 159; Sholly, Blum, and Smith, Ibid., 1451; Luttinger, Ibid., 1461; Rosenow and 
Osterberg, Ibid., 1919, LXXIII, 87. 



8l2 DIAGNOSTIC METHODS 

As J this measure is of such importance to health and sanitary officers, as well 
as*to*physicians in general, I give, herewith, some references for consultation.^ 

BIBLIOGRAPHY 

1. Besson. Practical Bacteriology, Microbiology and Serum Therapy. London, 

1913. 

2. Hewlett. A manual of Bacteriology. St. Louis, 1914. 

3. Irons. BiUings-Forchheimer Therapeusis, vol, 5. New York, 1914. 

4. Jordan. General Bacteriology. Philadelphia, 1913. 

5. KoLLE AND Hetsch, Die Experimentelle Bakteriologie und die Infektions- 

krankheiten. Berlin, 191 1. 

6. KoLLE AND Wassermann. Handbuch der Pathogenen Mikroorganismen. 

Jena, 1912 and 1913. 

7. Kraus and Levaditi. Handbuch der Technik und Methodik der Immuni- 

tatsforschung. Jena, 1909- 19 13. 

8. McFarland. Text-book on Pathogenic Bacteria. Philadelphia, 1913. 

9. Park and Williams. Pathogenic Microorganisms. Philadelphia, 1914. 

General Clinical Diagnosis 

1. Boston. A Text-book of Clinical Diagnosis. Philadelphia, 1905. 

2. Brown and Ritchie. Medical Diagnosis. Edinburgh, 1906. 

3. Brugsch and Schittenhelm. Lehrbuch klinischer Untersuchungsmethoden 

fiir Studierende und Aerzte. Berlin, 19 11. Technik der Speziellen 
Klinischen Untersuchungsmethoden. Berlin, 1914. 

4. Butler. Diagnostics of Internal Medicine. New York, 1905. 

5. Da Costa. Medical Diagnosis. Philadelphia, 1890. 

6. Debove et Achard. Manuel de diagnostic medical. Paris, 1900. 

7. Deguy et Guillaumin. Microscopie clinique. Paris, 19 12. 

1 See Wright, Lancet, 1896, II, 807; Brit. Med. Jour., 1897, 1, i6;Leishman, Jour. Roy. 
Inst. Pub. Health, 1910, XVIII, 394; Firth, Jour. Roy. Army Med. Corps, 191 1, XVII, 
495; Russell, Boston, Med. and Surg. Jour., 1911, CLXIV, i; Nelson and Hall, Jour. Am. 
Med. Assn., 1911, LVII, 1759; Schereschewsky, Pub. Health Rep., 1911, XXVI, 1508; 
Davis, Jour. Am. Med. Assn., 1912, LVIII, 537; Russell, Ibid., 1331; Maverick, Ibid., 
1672; Engelbach, Interstate Med. Jour., 1912, XIX, 537; Albert and Mendenhall, Am. 
Jour. Med. Sc, 1912, CXLIII, 232; Williams, Ibid., 352; Callison, Ibid., 1912, CXLIV, 
350; Russell, Bull. 2, War Dept. OfiSce Surg. Gen., Washington, 1913, 7; Craig, Ibid., 
15; Nichols, Ibid., 23; Hunt, Am. Jour. Med. Sc, 1913, CXLV, 826; Hiss, Jour. Med, 
Research, 1913, XXVIII, 385; Russell, Jour. Am. Med. Assn., 1913, LXI, 666; Metchnikoff 
and Besredka, Ann. de ITnst. Pasteur, 1913, XXVII, 597; Russell, Am. Jour. Med. Sc, 

1913, CXLVI, 803; Foxworthy, Military Surg., 1914, XXXIV, 332; Sachs, Med. Klin., 

1914, X, 1538; Russell, Jour. Am. Med. Assn., 1914; LXII, 1371; Krumbhaar and Richard- 
son, Am. Jour. Med. Sc, 1915, CXLIX, 406; Thomsen, Hospitals tid., 1915, LVIII, 211; 
Stursberg and Klose, Miinch. med. Wchnschr., 191 5, LXII, 380; Cecil, Jour. Infect. 
Dis., 1915, XVI, 26; Biedl, Wien. klin. Wchnschr., 1915, XXVII, 125; Paltauf, Ibid., 
125; Kirschbaum, Ibid., 208; Eggerth, Ibid., 209; Csernel, Marton and Feistmantel, 
Ibid., 22q; Sladek and St. Kotlowski, Ibid., 389; Goldscheider and Aust, Deutsch. med. 
Wchnschr., 191 5, XLI, 361; Kisskalt, Ibid., 393; Schneider, Ibid., 393; Reibmayr, Miinch. 
med. Wchnschr,, 1915, LXII; 610; Rhein, Ibid., 427; Bourke, Evans and Rowland, Brit. 
Med. Jour., 1915, I, 584; Widal, Bull, de FAcad. Med., 1915, LXXIV, 205; Vincent and 
Chantemesse, Ibid., 225; Harris and Ogan, Jour. Am. Med. Assn., 1915, LXIV, 3; Garbat, 
Ibid., 489; Trowbridge, Finkle and Barnard, Ibid., 728; Elmer, Ibid., 1147; Lyster, Ibid., 

1915, LXV, 510; Jobling and Petersen, Ibid., 515; Sawyer, Ibid., 1413; Schottstaedt, Ibid., 
1713; Nichols, Jour. Exper. Med., 1915, XXII, 780; Holler, Ztschr. klin. Med., 1915, 
LXXXI, 462; Noack, Ibid., LXXXII, 132; Stieve, Deutsch. Arch. f. klin. Med., 1915, 
CXVII, 462; Mertz, Ztschr. f. exp. Path. u. Therap., 1915, XVII, 224; Howell, Jour. In- 
fect. Dis., 1916, XIX, 63; Courmont and Devic, C. R. soc biol. Paris, 1916, CLXIII, 534; 
Mendelson, Mil. Surg., 1916, XXXIX, 361; Weston, Jour. A. M. A., 1916, LXVI, 1089; 
MUler, 111. Med. Jour., 1916, XXIX, 8; McCoy, Jour. A. M. A., 1917, LXVIII, 1401; 
Davison, Jour. Lab, and Chn. Med., 1917, II, 607; Russell, Jour. A. M. A., igig, LXXIII, 
1863. 



BIBLIOGRAPHY 813 

8. Emerson. Clinical Diagnosis. Philadelphia, 1913. 

9. French. Medical Laboratory Methods and Tests. Chicago, 19 12. 

10. GuiART ET Grimbert. Precis de diagnostic. Paris, 19 12. 

11. Greene. Medical Diagnosis. Philadelphia, 1907. 

12. Hare. Practical Diagnosis. Philadelphia, 1908. 

13. Hutchinson and Rainy. Clinical Methods. Chicago, 1908. 

14. Jagic. Klinische Mikroskopie. Wien, 1913. 

15. KoRANYi UND RiCHTER. Physikalische Chemie und Medizin. Leipzig, 1908. 

16. VoN Jaksch. Klinische Diagnostik. Berlin, 19 10. 

17. Klemperer. Grundriss der klinischen Diagnostik. Berlin, 1906. 

18. Kraft. Analyt. Diagnostikum. Leipzig, 19 13. 

19. Lenhartz. Mikroskopie und Chemie am Krankenbett. Berlin, 19 13. 

20. Leube. Specielle Diagnose der inneren Krankheiten. Leipzig, 1908. 

21. Morris. Clinical Laboratory Methods. London, 1913. 

22. MussER. Medical Diagnosis. Philadelphia, 1904. 

23. Von Noorden. Handbuch der Pathologic des Stoffwechsels. Berlin, 1906. 

24. Panton. Clinical Pathology. London, 19 13. 

25. Sahli. Lehrbuch der klinischen Untersuchungsmethoden. Wien, 19 13. 

26. Schilling. Aerztliche Technik. Wiirzburg, 1906. 

27. Simon. A Manual of Clinical Diagnosis. Philadelphia, 1914. 

28. Todd. Manual of CHnical Diagnosis. Philadelphia, 1912. 

29. Wesener. Medizinisch-klinisch Diagnostik. Berlin, 1907. 

30. Wood. Chemical and Microscopical Diagnosis. New York, 191 1. 

31. Zuelzer. Chemische und Mikroskopische Diagnostik. Leipzig, 1906. 



INDEX 



Abderhalden's sero-diagnosis of cancer, 
716 

of dementia praecox, 717 

of pregnancy, 703 
Abortion, 406 
Abscess, blood in, 603 

indican in, 271 

of liver, sputum in, 34 

of lung, sputum in, 34 
Absorptive power of stomach, 86 
Acanthia lectularia, 167 
Acarus scabiei, 165 
Accidental albuminuria, 275 
Acetic acid in gastric contents, 64, 73, 

11 
Aceto-acetic acid, 196, 341 
Acetone bodies, 196, 334 

in blood, 502 

in urine, 196, 334 

determination of, 339 
significance of, 334 
tests for, 338 
Acetonemia, 502 
Acetonuria, 334 
Acholic stools, 107, 116 
Achorion Schonleinii, 168 
Achroiocythemia, 465 
Achromatophilia, 552 
Achioo dextrin, 36, 82 
Achylia gastrica, 78, 89 
Acid, acetic, 64, 73, 77, 262 

aceto-acetic, 196, 341 

alloxyproteic, 213, 261 

amino, 82, 196, 259 

bile, 61, 109, 349 

butyric, 64, 73, 76, 262 

cholalic, 117, 196 

chondroitin-sulphuric, 196, 213, 266 

diacetic, 196, 341 

diamino, 83, 92, 259 

fatty, 13, 115, 123, 196, 262, 498 

formic, 262 

glycocholic, 117, 196, 349 

glycosuric, 351 

glycuronic, 196, 331 

hippuric, 196, 261, 363 

homogentisic, 196, 351 

hydrochloric, 51, 65 

hydroquinone-acetic, 351 

lactic, 64, 73, 74, 262 

nucleinic, 245, 266 

oxalic, 196, 263 

oxaluric, 196, 263 

/3-oxybutyric, 196, 343 

oxymandelic, 196 

oxyproteic, 196, 213, 261 

phosphoric, 196, 203 



Acid, picric, 287 

propionic, 262 

rosacic, 268 

rosolic, 21 

sulphuric, 211 

taurocholic, 117, 196, 349 

uric, 196, 242, 356, 481 

uroferric, 213 

uroleucic, 196, 351 
Acid-fast organisms, 20 
Acidity of gastric juice, 64 

of urine, 187, 190 
Acidophilic cells, 552 

granules, 552, 559 
Acidosis, 189, 334, 419 
Acid stains, 532 

unit, 191 
Actinomyces in sputum, 27 
Actinomycosis, 27 
Acute anterior poliomyelitis, 760 

bronchitis, sputum in, 31 

gastritis, gastric juice in, 90 

hemorrhage, anemia due to, 592 

infectious conjunctivitis, 47 
diseases, blood in, 606 

leukemia, 600 

nephritis, 183, 185, 194, 199, 206, 
222, 275 

rheumatism, blood in, 614 

yellow atrophy of the liver, urine 
in, 225, 259, 361 
Addison's disease, blood in, 606 
Adenin, 252 

Adler's test for blood, no 
Adolescent albuminuria, 278 
Aerobic cultures, 786 

organisms, 786 
^stivo-autumnal malaria, 624, 627 
Agar, ameba, 783 

ascitic, 784 

blood, 783 

dextrose, 783 

glycerin, 783 

hydrocele, 784 

litmus lactose, 783 

nutrient, 782 
Age, effect of, on red blood cells, 548 

on white blood cells, 578 
Agglutination, 660, 669 
Agglutinins, 660 
Agglutinophore, 660 
Agonal leucocytosis, 572 
Air in sputum, 4 
Albumin, determination of, 286 

in exudates, 737 

in feces, 122 

in milk, 771 



815 



8i6 



INDEX 



Albumin, in sputum, 5 
Albumin, in transudates, 737 

in urine, 274 

of blood, 466 

quotient, 289, 468 

'removal of, 288 

serum, 274 

significance of, in urine, 274 

tests for, 280 
Albuminuria, accidental, 275 

adolescent, 278 

after baths, 276 

alimentary, 276 

colliquative, 279 

constitutional, 277 

cyclic, 277 

false, 275 

febrile, 278 

functional, 275 

hematogenous, 279 

hypostatic, 278 

intermittent, 277 

lordotic, 277 

mixed, 295 

neurotic, 279 

of the new-born, 276 

of pregnancy, 276 

orthostatic, 277 

orthotic, 277 

physiologic, 276 

post-infectious, 278 

postural, 277 

renal, 279 

structural, 278 

thermolytic, 292 

toxic, 279 

traumatic, 279 

true, 275 

vesicular, 275 

with definite renal lesions, 279 
Albumon, 466 
Albumose, Bence-Jones, 290 

in blood, 468 

in feces, 122 

in gastric contents, 82 

in urine, 293 
Albumosuria, 293 

alimentary, 294 

digestive, 294 

enterogenous, 294 

febrile, 294 

hematogenous, 294 

hepatogenous, 294 

myelopathic, 291 

pyogenic, 294 

renal, 295 

significance of, 294 

tests for, 293 
Alcohol-fast organisms, 20 
Aleukemic myelosis, 601 
Alimentary albuminuria, 276 

albumosuria, 294 

chloruria, 198 

galactosuria, 322, 329 

glycosuria, 299 

levulosuria, 322 

lipuria, 364 

pentosuria, 325 
Alkaline phosphates, 204 



Alkaline reserve of blood, 436 

tide of urine, 191 
Alkalinity of blood, 418 
Alkalinuria, 205 
Alkaptonuria, 350 
Allantoin, 196, 261 
Allergy, 662 

Alloxur bases, 122, 252, 359 
Alloxyproteic acid, 196, 213, 261 
Almen-Nylander's test for glucose, 308 
Almen's tannic acid solution, 770 
Aloin test for blood, 109, 729 
Altitude, effect of, on red cells, 549 
Alveolar epithelial cells in sputum, 1 1 
Ambard's coefficient, 477 
Amblyochromatic erythroblasts, 546 
Amboceptors, 659, 687, 694 
Amboceptor paper, 694 
Ameba agar, 783 

classification of, 135 

cysts of, 138 
Ameba coli in feces, 135 

in mouth, 42 

in sputum, 29 

in urine, 384 

pulmonalis, 29 
Amebic dysentery, 135 

pyorrhea, 42 
American hook-worm, 155 
Amino-acetic acid, 259 
.Amino acids in urine, 196, 259 
a-aminoisobutyl-acetic acid, 361 
Ammonia in blood, 487 

in urine, 237 
Ammoniemia, 487 

Ammonium magnesium phosphate in 
feces, 120 
in sputum, 13 
in urine, 366 

urate calculi, 386 
sediment, 365 
Amniotic fluid, 405 
Amount of blood, 410 

of cerebrospinal fluid, 756 

of feces, 104 

of gastric juice, 57 

of sputum, 2 

of urine, 180 
Amyloid kidney, globulin in urine of, 
289 

urine in, 183, 186, 280, 289 
Amylopsin, 102 
Amylosis, 34 
Anachlorhydria, 70 
Anaerobic cultures, 786 

organisms, 786 
Anaphylaxis, 808 
Ancylostoma duodenale, 154 
Ancylostomiasis, 154 
Andrade's indicator, 132 
Anemia, 585 

aplastic, 591 

Biermer's, 587 

chlorotic, 585 

definition of, 585 

EhrHch's, 591 

due to acute hemorrhage, 592 
to acute infections, 595 
to bad air, 594 



INDEX 



817 



Anemia, to blood poisons, 595 
to chronic disease, 615 
to chronic hemorrhage, 593 
to inanition, 593 
to intestinal parasites, 594 
Anemia, febrile, 595 

hemolytic, 595 

infantum pseudoleukemica, 590 

von Jaksch's, 590 

leukanemia, 590 

lymphatic, 590 

of the South, 155 

of the tropics, 548 

primary pernicious, 587 

progressive pernicious, 587 

secondary, 591 

simple primary, 585 

splenic, 589 
Anemic degeneration, 552 
Anesthesia, changes in urine after, 336 

effects of, on blood, 604 
Angioneurotic hematuria, 373 
Anguillula aceti in urine, 385 

intestinalis et stercoralis, 150 
Anhydremia, 413 
Animal gum in urine, 331 
Animal parasites in blood, 619 

in ear, 47 

in feces, 133 

in gastric contents, 63 

in sputum, 28 

in urine, 384 
Ankylostomum duodenale, 154 
Anopheles Crucians, 619 

maculipennis, 619 
punctipennis, 619 
quadrimaculatus, 619 
Anterior urethritis, 371 
Anthracosis, 4, 34 
Anti;5mboceptors, 659 
Antibodies, 656, 680, 686 
Anticcmolement, 659 
Antiformin method for tubercle bacilli, 

18 
Antigens, 656, 681, 683, 689, 696 
Antihemolysins, 659 
Antihuman amboceptor, 694 
Antimeningoccic serum, 759 
Antisheep amboceptor, 687 
Antitoxins, 656 

Antityphoid vaccination, 676, 811, 
Anuria, 183 
Aplastic anemia, 591 
Appearance of blood, 416 

of exudates, 738 

of feces, 105 

of gastric contents, 57 

of leucocytes, 557 

of milk, 768 

of red cells, 541 

of semen, 398 

of spinal fluid, 756 

of sputum, 4 

transudates, 737 

of urine, 183 
Appetite juice, 51 
Arabinose, 325 
Arginin, 260 
Arneth's classification of neutrophils, 561 



Arnold's test for diacetic acid, 342 

Arnold- Volhard method for chlorids, 200 

Arterial blood, 417 

Arthropoda, 164 

Ascaridae in feces, 148 

Ascaris alata, 149 

Ascaris caniculae, 146 

canis, 149 

canis et martis, 163 

cati, 144 

felis, 149 

grsecorum, 149 

lumbricoides, 148 

lumbricus canis, 149 

marginata, 149 

mystax, 149 

teres, 149 

trichiura, 152 

tricuspidata, 149 

vermicularis, 149 

visceralis et renalis, 163 

werneri, 149 
Ascitic agar, 784 

fluid, cytology of, 751 
Asexual cycle of malarial parasite, 622 
Ash-free diet of Taylor, 200 
Asiatic cholera, feces m, 130 

organism of, 129 
Aspergillus flavus, 16 

fumigatus, 16 

in aural secretion, 47 

in sputum, 16 

niger, 16 

subfuscus, 16 
Assimilation limit, 301 
Asthma, bronchial, 33 

eosinophilia in, 10, 33, 576 

sputum in, 33 

fusiform bacillus in, 33 
Aural secretion, 46 

larvae in, 47 

molds in, 47 
Autotoxic enterogenous cyanosis, 551 
Autovaccines, 661, 811 
Azoospermatism, 400 
Azotorrhea, 114 

Babcock's method for fat in milk, 772 
Bacillary dysentery, 132 

index, 661 
Bacilluria, 384 
Bacillus aerogenes capculatus, 133, 797 

anthracis, 26 

coli communis, 48, 129, 384, 650, 

793 
comma, 61, 129, 799 
Ducrey's, 744 
euteritidis sporogenes, 798 
hodgkini, 601, 791 
icteroides, 641 
lepras, 24 
mallei, 28 

mucosus capsulatus, 25, 46 
of Boas-Oppler, 62 
of Bordet-Gengou, 26, 791 
of bubonic plague, 27 
of diptheria, 38, 789 
of dysentery, 132, 795 
of Finkler-Prior, 61, 130 



8i8 



INDEX 



Bacillus aerogenes capsulatus, of Fried- 
lander, 25, 46 
of glanders, 28 
of Hansen, 24 
of Hoffman, 40, 790 
of influenza, 26, 791 
of Kitasato and Yersin, 27 
of Klebs-Loffler, 38, 789 
of Koch, 18, 132, 389, 654, 741 
of Koch- Weeks, 48 
of Morax-Axenfeld, 48 
of ozena, 46 
of Perez, 46 
of Pfeiffer, 26, 33, 791 
of Sanarelli, 641 
of Shiga, 132, 795 
of soft chancre, 744 
of tuberculosis, 17, 132, 389, 654, 

741 

of Tunnicliff, 45 

of Vincent, 40 

of Welch, 133, 797 

of whooping-cough, 26, 791 

paratyphoid, 131, 649 

perfringens, 798 

pertussis, 26, 791 

pestis, 27 

pseudo-diphtheria, 40, 790 

pyocyaneus, 4 

rhinitis, 45 

smegma, 24, 364, 743 

timothy, 24 

typhosus, 26, 130, 384, 792 

ulceris cancrosi, 744 

xerosis, 40 

X of Sternberg, 641 
Bacteria in blood, 649 

in conjunctiva, 47 

in ear, 46 

in exudates, 741 

in feces, 128 

in gastric contents, 62 

in milk, 775 

in mouth, 36 

in nasal secretions, 44 

in sputum, 14 

in urine, 381 
Bacterial flora of feces, 129 

of vagina, 402 
Bacterial vaccines, 661, 808 
Bacteriema, 649 
Bacteriology of blood, 649 

of cerebrospinal fluid, 759 

of exudates, 741 

of feces, 128 

of milk, 775 

of sputum, 14 

of tirine, 381 
Bacteriolysins, 658 
Bacteriuria, 384 

Balantis, erosive and gangrenous, 744 
Balantidium coli, 141 
Baldwin's method for oxalic acid, 

264 
Bang's test for albumose, 293 

for sugar, 313 
Banti's disease, blood in, 589 
Barberio's test for semen, 402 
Barber's itch, organism of, 169 



Bases, alloxur, 122, 196, 252, 359 
hexone, 83, 92, 259 
nuclein, 122, 196, 253, 360 
purin, 122, 196, 359 
xanthin, 122, 196, 252, 359 
Basic stains, 532 
Basket cells, 565 
Basophile leucocytes, 562 
Basophiles, 562 
Basophiha, 553, 562 

Basophilic degeneration of red cells, 553 
Basophilic stippling of reds, 554 
Bass and Watkin's macroscopic agglu- 
tination test, 674 
Baths, albuminuria following, 276 

effect of, on red cells, 550 
Beckmann apparatus, 444 
Bed bug, 167 
Beef tape- worm, 143 
Bence- Jones, body, 290 
protein, 290 
amount of, 291 
significance of, 291 
tests for, 292 
Benedict and Murlin's test for amino 

acids in urine, 260 
Benedict's test for sugar, 305 
for uric acid in blood, 481 
Benzi din test for blood, no 
Benzoic acid, 261 
Bial's test for pentose, 327 
Biermer's anemia, 587 
Bile acids in blood, 502 
in feces, 117 
in gastric contents, 61 
in urine, 196, 349 
media, 793 

pigments in blood, 502 
in exudates, 738 
in feces, 108, 117 
in gastric contents, 59, 61 
in sputum, 3 
in urine, 196, 346 
significance of, 346 
tests for, 347 
Bilharzia hematobia, 29, 646 
Bilharziasis, 646 
Bilicyanin, 117 
Bilifusein, 117, 347 
Bilihumin, 117 
Biliprasin, 108, 347 
Bilirubin, 59, 108, 117, 347, 363 
Biliverdin, 59, 108, 117, 347 
Biologic test for blood, 677 
Bismuth oxid in stools, 107 

test for glucose, 308 
Biuret test for protein, 292 
Black's method for /i-oxybutync acid, 

344 
Black sputum, 4 

urine, 186 

-water fever, 296 
Bladder, inflammation of, 191, 370 

tuberculosis of, 383 
Blastomycetes in skin, 172 

in sputum, 17 
Blastomycosis, 17, 172 
Blennorrhea, 403 j 
Blood, 408 



INDEX 



819 



Blood, acetone in, 502 
after anesthesia, 604 
after splenectomy, 618 
after surgical intervention, 603 
agar, 783 
albumin in, 466 
alkalinity of, 418 
amino acids, in, 492 
ammonia in, 487 
bacteriology of, 646 
biliary constituents in, 502 
carbohydrates in, 494 
casts J 377 
cells in, 509 

chemical properties of, 446 
chemical tests for, 109, 728 
cholesterin in, 499 
coagulation of, 440 
color-index of, 465 
color of, 417 
constituents of, 417 
counting of cells of, 509, 515, 566 
creatinin in, 490 
crises, 546 
cryoscopy of, 443 
cultures, 646 
dust, 581 

electric conductivity of, 444 
enumeration of cells of, 509, 515, 566 
fat in, 498 
ferments of, 509 
fixation of smears of, 528 
formation of, 409 
fresh, 524 
gases in, 508 
glucose in, 494 
grouping of, 724 
hemoglobin of, 448 
in abscess formation, 603 
in acute infections, 606 
in acute rheumatism, 614 
in Addison's disease, 606 
in aplastic anemia, 591 
in bilharziasis, 646 
in carcinoma, 616 
in chlorosis, 585 
in chronic diseases, 615 
in chronic tuberculosis, 615 
in diabetes mellitus, 604 
in diphtheria, 613 
in distomiasis, 646 
in filariasis, 634 
in gout, 605 
in kala-azar, 634 
in leprosy, 616 
in leukanemia, 590 
in leukemia, 596 
in malaria, 619 
in measles, 611 
in myxedema, 606 
inorganic constituents of, 503 
in pernicious anemia, 587 
in pertussis, 613 
in pneumonia, 607 
in primary anemia, 585 
in pseudoleukemia, 601 
in relapsing fever, 63 1 
in rickets, 606 
in Rocky Mountain spotted fever, 645 



Blood, in scarlet fever, 610 
in secondary anemia, 591 
in sleeping sickness, 632 
in splenic anemia, 589 
in syphilis, 615, 636 
in typhoid fever, 609, 648 
in varicella, 613 
in variola, 612 
in whooping-cough, 613 
in yellow fever, 641 
limitations of examinations of, 732 
medico-legal aspects of, 727 

tests for, 727 
morphology of, 524 
needle, 415 
nitrogen of, 469 
non-protein nitrogen of, 469 
obtaining of, 415 
odor of, 418 

osmotic pressure of, 443 
parasitology of, 619 
pathology of, 584 

general, 603 

special, 584 
physiology of, 409 
pigments of, 448 
plates, 523, 580 
properties of, 416 
proteins of, 466 
reaction of, 418 
red cells of, 541 
serum reactions of, 669 
smears of, 526 
solids of, 448 
specific gravity of, 437 
spectroscopic tests for, 449, 732 
staining of smears of, 533 
tests for, 109, 728 
total solids of, 448 
urea in, 474 
uric acid in, 48 1 
value of examinations of, 732 
viscosity of, 439 
vital staining of, 540 
volume of, 410 

volume relations of elements of, 413 
white cells of, 557 
Blood casts, 377 

cells in exudates, 739 

in feces, 120 

in gastric contents, 61 

in sputum, 3, 5, 11 

in urine, 346, 372 
plates, 580 

appearance of, 580 

counting of, 523 

function of, 581 

number of, 580 

size of, 580 

staining properties of, 581 
poisons, 595 
smears, fixation of, 528 

preparation of, 526 

staining of, 531 
staining, 531 
Bloody sputum, 3, 5, 11 
Bloor's method for cholesterin, 500 
Bloor and Knudson's method for choles- 
terin esters, 502 



820 



INDEX 



Boas' method for lactic acid, 76 

for estimating gastric motility, 85 

test for free hydrochloric acid, 67 

test-meal, 55 
Boas-Oppler bacillus, 62 
Body louse, 166 
Boggs' coagulometer, 442 

method for protein in milk, 770 
Bone-marrow, 409, 582 

function of, 409 

morphology of, 582 
Borax as preservative in milk, 775 
Bordet's potato medium, 791 
Boric acid as preservative, 775 
Bothriocephaloidea in feces, 146 

bothriocephalus latissimus, 146 

bothriocephalus latus, 146 

bothriocephalus sp. Ijima et Kuri- 
moto, 147 

dibothriocephalus cordatus, 147 
Bottcher's cr3^stals, 14, 398 
Bouillon, 780" 
Bradshaw's myelopathic albumosuria, 

291 
Breakfast, test, 54 
Bremer's blood-test in diabetes, 604 
Brem's method for grouping blood, 726 
Bricklayers' anemia, 156 
Brilliant green in culture media, 131 
Brodie-Russell coagulometer, 442 
Bronchial asthma, 33 

stones, 8 
Bronchioliths, 8 
Bronchiolitis exudativa, 7 
Bronchitis, acute, 31 

chronic, 32 

eosinophilic, 10 

fetid, 32 

fibrinous, 32 

putrid, 32 
Broncho-pneumonia, sputum of, 31 
Bronfenbrenner's modification of Abder- 

halden test, 713 
Bruck's test, 718 

Bunge-Trantenroth's method for tuber- 
cle bacilli, 24 
Burri's method for spirochaete, 745 
Busk's intestinal fluke, 161 
Butyric acid in gastric contents, 64, 73, 
11 

Cabot's ring bodies, 554 
Cachexial fever, 634 
Cadaverin, 123 
Caffein, 253 

Calcium carbonate calculi, 388 
sediment, 367 
of urine, 216 ; 
oxalate calculi, 386 
crystals in sputum, 1 1 
in urine, 359 
phosphate calculi, 388 

sediment, 365, 366 
soaps in feces, 120 
sulphate sediment, 363 
Calculi, ammonium urate, 386 
biliary, 117 
bronchial, 8 
calcium carbonate, 388 



Calculi, calcium, oxalate, 386 

classification of, 385 

cystin, 388 

examination of, 387 

formation of, 385 

hepatic, 117 

intestinal, 118 

nasal, 46 

phosphatic, 388 

pulmonary, 8 

renal, 385 

table for examination of, 387 

ureteral, 385 

urethral, 385 

uric acid, 386 

urostealith, 388 

vesical, 385 

xanthin, 388 
Calmette's tuberculin reaction, 664 
Cammidge's reaction, 328 
Cancer (see Carcinoma) 
Cane sugar, absorption of, 123 

digestion of, 64 

in urine, 331 
Capsule, staining of, 652 
Carbohydrates, digestion of, 36, 64, 82 

in blood, 493 

in exudates, 738 

in feces, 116, 123 

in milk, 773 

in urine, 298 
Carbol-fuchsin solution, 20 
Carbonates in urine, 216 
Carbon dioxide capacity of blood. Van 
Slyke and CuUen's Method for, 
422 

hemoglobin, 451 
in blood, 451 

monoxid hemoglobin, 451 
poisoning, 451 
Carcinoma, Abderhalden's test in, 716 

blood in, 616 

cells in exudates, 751 

fragments in feces, 118 
in gastric contents, 63, 93 
in urine, 381 

of cervix, 407 

of kidney, 372, 381 

of pleura, 751 

of rectum, 118 

of stomach, 91 

of uterus, 407 
Cardiac albuminuria, 277 

edema, 182 

pleurisy, 751 
Carnin, 252 
Casein in feces, 115 

appearance of, 115 
Leiner's test for, 115 

in milk, 771 
Casts, 374 

blood, 377 

chemistry of, 374 

colloid, 378 

epithelial, 377 

fatty, 377 

fibrinous, 377 
in sputum, 8 

granular, 375 



INDEX 



821 



Casts, hyaline, 374 

mixed, 374 

origin of, 374 

prostatic, 400 

pseudo, 379 

pus, 377 

significance of, 375, 379 

size of, 374 

staining of, 375 

testicular, 400 

true, 374 

waxy, 376 
Catalase, 579 
Catarrhal stomatitis, 38 
Cellulose in feces, 117 
Centrifugation, 354 
Cercomonads in feces, 140 

in sputum, 28 

in urine, 385 

in vaginal secretions, 404 
Cercomonas coli hominis, 140 

hominis, 140 

intestinalis, 140 

seu Bodo urinarius, 140 
Cerebrospinal fluid, 754 

bacteriology of, 759 

chemistry of, 757 

cytology of, 758 

in nasal secretion, 45 

microscopy of, 758 

obtaining of, 755 

pressure of, 755 

properties of, 756 

syphilis, 761 
Cerumen, 46 
Cestodes in feces, 141 

in sputum, 29 
Chalicosis, 4, 34 
Chancre, organism of, 744 
Character of blood, 416 

of exudates, 738 

of feces, 105 

of sputum, 4 

of urine, 180 
Charcoal in feces, loi 
Charcot-Leyden crystals in feces, 120 

in sputum, 14, 30, 31 
Cheesy masses in sputum, 6 
Chemical fixation of smears, 530 
Chemotaxis, 660 
Childhood, red cells in, 556 

white cells in, 578 
Chinese liver-fluke, 162 
Chinovose, 325 
Chloremia, 465 
Chlorid excretion in urine, 197, 391 

retention, 197 
Chlorids of the blood, 503 

of the plasma, 503 

of the urine, 197 
amount of, 197 
estimation of, 250 
variations of, 197 
Chloromata, 4 
Chlorosis, 585 
Chlorotic anemia, 585 
Chloruria, 197, 391 
Cholecyanin in feces, 107 

in urine, 347 



Chololil hiasis, 117, 499 

Cholemia, 502 

Cholera red reaction, 800 

spirillum, characteristics of, 799 
in feces, 129 
Cholesterin in blood, 499 

crystals in feces, 120 

in sputum, 13 

in urine, 364 
Choletelin in urine, 347 
Choluria, 346 
Chondroitin-sulphuric acid, 196, 213, 

266 
Chromogenic bacteria in sputum, 4 
Chromogens in urine, 267 
Chronic bronchitis, sputum in, 32 

diseases, blood in, 604, 615 

gastritis, gastric juice in, 90 

nephritis, urine in, 183, 185, 194, 
199, 206, 279 
Chyloid exudates, 739 
Chylous exudates, 739 
Chyluria, 185, 364 
Chymosin, 64, 81 
Cimaenomonas hominis, 140 
Cimex lectularius, 167 
Cladocoelium hepaticum, 160. 
Clark's method for elastic tissue, 12 
Clay-colored stools, 107, 117 
Cleaning glass-ware, 524 
Clearing of urine, 317 
Coagulation of blood, 440 

of exudates, 738 

of milk, 769 

of urine, 287 

time of blood, 440 
Coagulometer of Boggs, 442 

of Dorrance, 441 

of Rudolf, 441 

of Russell-Brodie, 442 
Coagulo reaction for syphilis, 719 
Coal pigment in sputum, 4, 34 
Coarsely granular cells of Schultze, 562 
Coating of the tongue, 38 
Coccidioidal granuloma, 173 
Coccidioides immitis, 173 
Coccidium hominis, 139 

perforans, 139 
Coccobacillus foetidus ozen^, 46 
Coefficient, creatinin, 254 

of Ambard, 477 

of Haeser, 194 

of Haines, 194 

of Long, 194 

refraction, 467 
Colitis, catarrhal, 116 

malignant, 118 

mucous, 112 
Collection of feces, 99 

of gastric contents, 52 

of puncture fluids, 737 

of sputum, 2 

of urine, 179 
Collignon and Pilod's test, 760 
Colloid casts, 378 

cysts, 752 
Colloidal nitrogen, 261 
Colon bacillus, 48, 129, 384, 650, 793 
Color-index of blood cells, 465 



822 



INDEX 



Color-index of blood, 417 

of exudates, 738 

of feces, 106 

of gastric contents, 58 

of sputum, 3 

of urine, 184 
Colorimeter, 257 

Colorimetric method for H ion concen- 
tration, 429 
Colostrum, 766 
Coma diabeticum, 337 
Combined hydrochloric acid, 71 
Comma bacillus of Koch, 61, I29,[799 
Common flea, 168 

hver-fluke, 160 
Complement, 659, 688, 694 

fixation test, 680, 701 

preservation of, 688 
Complementophile, 659 
Composition of blood, '_446 

of milk, 767 

of urine, 195 
Concretions, biliary, 117 

in sputum, 8 

in bronchioliths, 8 

in pneumoliths, 8 
intestinal, 118 
coproliths, 118 
enteroliths, it8 . 

nasal, 46 

renal, 385 

vesical, 385 
Conductivity, electric, of blood, 445 

of urine, 390 
Congo-red test, 66 
Conjugated glycuronic acids, 332 
Conjunctival secretions, 47 
Conjunctivitis, diphtheritic, 48 

gonorrheal, 48 

infectious, 48 

vernal, 49 
Consistency of blood, 439 

of feces, 105 

of gastric contents, 59 

of milk, 768 

of spinal fluid, 756 

of sputum, 2 

of urine, 183 
Constipation, 105 
Coproliths, 118 
Corpora amylacea, 399 
Corynebacterium granulomatis maligni, 

791 
Cough, whooping, blood in, 613 

organism of, 26, 791 
Counting of blood plates, 523 

of pus cells, 372 

of red cells, 509 

of white cells, 520 
Crab louse, 166 
Creatin in blood, 492 

in urine, 253, 258 
Creatinin coefficient, 254 

estimation of, 256, 491 

metabolism of, 253, 490 

tests for, 255 

variations of, 253, 490 
Creatorrhoea, 114 
Crenation, 542 



Crescents in blood, 627 
Crises, blood, 546 
Cryoscopy of blood, 443 

of urine, 390 
Crystals in feces, 120 

in gastric contents, 63 

in semen, 398 

in sputum, 13 

in urine, 356 
Csonka's method for acetone, 339 
Culex mosquito, 641 
Cultures, aerobic, 786 

anaerobic, 786 

blood, 646 

brilliant green in, 131 

media, 780 

throat, 39 

urine, 381 
Curds in feces, 115 
Curschmann's spirals, 7, 30, 31 
Cutaneous reactions, 662 
Cyclic albuminuria, 277 
Cylindroids, 378 
Cylindruria, 379 
Cyst, colloid, 752 

dermoid, 753 

fluids, 752 

hydatid, 754 

hydrocele, 753 

hydronephrotic, 754 

myxoid, 752 

ovarian 752 

pancreatic, 754 

papillary, 753 

parovarian, 753 

serous, 752 

spermatocele, 754 
Cystein, 213 

Cysticercus cellulosae, 143 
Cystin, 213 

calcuh, 388 

sediment, 360 
Cystinuria, 360 
Cystitis, 191, 370, 383 
Cystorpermium hominis, 139 
Cystotaenia solium, 143 
Cytology in cardiac pleurisy, 751 

in malignant pleiirisy, 751 

in nephritic pleurisy, 751 

in pneumococcus pleurisy, 750 

in primary tubercular pleurisy, 750 

in secondary tubercular pleurisy, 750 

in streptococcus pleurisy, 750 

in typhoid pleurisy, 751 

of ascitic fluid, 751 

of cerebrospinal fluid, 758 

of exudates, 748 
of peritoneum, 751 
of pleura, 750 

of normal-fluids, 749 

of sputum, 10 

technic of, 748 
Cytophile, 656 

Dahlia stain, 563 
Daland's hematocrit, 413 
Dare's hemoglobinometer, 459 
Darling's method of staining amebas, 138 
Day urine, 181 



INDEX 



823 



Dechloridization, 199 

Deficit of hydrochloric acid, 73 

Definitive host, 143 

Degenerated forms of red cells, 553 

of white cells, 565 
Degeneration, anemic, 552 

hemoglobinemic, 555 
Degree of tolerance, 299 
Delayed chloroform poisoning, 336 
Wassermann reaction, 697 
Dementia praecox, Abderhalden's test in, 

717 
Demodex f olliculorum, 1 65 
Dermacentor Andersoni, 646 

venustus, 646 
Dermoid cysts, 753 
Desmoid bag, 87 
Deutero-albumose, 82, 293 
Dextrin in urine, 331 
Dextrose agar, 783 
Diabetes alternans, 245 

insipidus, 182 

mellitus, 182, 185, 300, 335 
blood in, 604 
Bremer's test in, 604 
lipemia in, 604 
urine in, 182, 185, 300, 335 
Williamson's test in, 605 

phosphatic, 206 

renal, 298 
Diabetic coma, 337 
Diacanthos polycephalus, 156 
Diacetic acid in urine, 341 
Diagnosis, functional, 388 
Diagnostic value of agglutination test, 

675 

of complement fixation test, 698 

of precipitin test, 679 
Diamines in urine, 123, 360 
Diamino acids, 83, 92, 259 
Diaminuria, 360 
Diarrhea, 105 
Diastase in urine, 265 
Diazo reaction, 352 
Dibothriocephalus cordatus, 147 

latus, 146 
Dibothrium latum, 146 
Diet of Folin, loi 

of Schmidt and Strasburger, 100 

of Taylor, 200 
Dieudonne's medium, 800 
Differential counting, 566 
Diffusible alkalinity of blood, 418 
Digestion, gastric, 82 

intestinal, 103, 121 

leucocytosis of, 569 

products of, 82, 103 
Digestive insufficiency, 113 
Dilatation of stomach, 83, 84 
Diluting fluids for blood, 514, 520 
Dimethylaminoazobenzol test, 66 
Dimethylaminobenzaldehyd reaction, 354 
Dimorphous muris, 140 
Diphtheria, bacillus of, 38, 789 

blood in, 613 

taking smear in, 39 
Diphtheritic conjunctivitis, 47 

laryngitis, 39 
Diplacanthus nana, 144 



Diplococcus cinereus, 805 

crassus, 805, 807 

flavus, 805, 807 

intracellularis meningitidis, 759, '806 

lanceolatus, 25, 31 

of Bonome, 759 

of Fraenkel, 25, 31, 651, 801 

of Jaeger and Heubner, 759 

of Neisser, 741, 805 

of Weichselbaum, 759, 806 

pneumoniae, 25, 31, 651, 801 
Diplogonoporus grandis, 147 
Diplomellituria, 302 
Dipylidium caninum, 144 

cucumerinum, 144 
Distoma capense, 646 

pulmonale, 29 

Ringeri, 29 

Westermanii, 29 
Distomiasis, 646 
Distomum buski, 161 

caviae, 160 

conus, 162 

crassum, 161 

hematobium, 30, 646 

hepaticum, 160 

hepatis endemicum seu perniciosum, 
162 

hepatis innocuum, 162 

japonicum, 162 

lanceolatum, 162 

sibiricum, 162 

sinense, 162 

spathulatum, 162 

tenuicolle, 162 
Ditrachyceros rudis, 156 
Dittrich's plugs, 6 
Dochmius ancylostomum, 154 

duodenalis, 154 
Dohle's inclusion bodies, 611 
Donaldson's method for staining ame- 
bic cysts, 138 
Donne's test for pus, 372 
Donogany's test for hemoglobin, 297 
Doremus ureometer, 230 
Dorrance's method for coagulation-time, 

441 
Drigalski and Conradi's media, 131 
Drop method for Wassermann test, 692 
Dropsical cells, 547, 586 
Dropsy of chorionic villi, 406 
Drugs, effects of, on blood, 468, 550 

reactions of, in urine, 186 
Dry test of feces for blood, 1 1 1 
Ducrey's bacillus, 744 
Durham's hemocytometer, 522 
Dum-dum fever, 634 
Dwarf tape-worm, 144 
Dysentery, amebic, 135 

bacillary, 132 
Dysmenorrhea, 405 
Dyspepsia, 91 

Earthy phosphates, 204, 206 

Eberth's bacillus, 26, 130, 384, 640, 792 

Echinococcus in feces, 145 

in sputum, 9 

in urine, 385 
Ectasis gastric, 85 



824 



INDEX 



Eel, vinegar, 385 
Effusions, pleuritic, 750 
Egg-yellow reaction, 353 
Eg}^tian chlorosis, 156 
Ehrlich's anemia, 591 

anemic degeneration, 552 

classification of leucocytes, 558 

dahlia stain, 563 

diazo reaction, 352 

dimethylaminobenzaldehyd reaction, 

354 

egg-yellow reaction, 353 

hemoglobinemic degeneration, 553 

side-chain theory, 656 

tri-acid stain, 535 

triple stain, 535 
Einhorn's method for total acidity, 72 

saccharometer, 320 
Elastic tissue in feces, 115 

in sputum, 12 
Electric conductivity of blood, 445 

of urine, 390 
Empyema, perforating, 34 
Endamoeba buccalis, 42 

coH, 139 

gingivalis, 42 

histolytica, 135 

tetragena, 136 
Endo medium, 132 
Endotheliosis, 751 
Enteritis, catarrhal, no 

malignant, 118 

membranous, 112 

mucous, 112 
Enterokinase, 102 
Enteroliths, 118 
Enthelmintha, 141 
Entozoa in feces, 141 
Enumeration of iDlood cells, 509, 520, 566 

of pus cells, 372 
Eosin-hematoxylin stain, 534 

methylene-blue stain, 47 
Eosinophiles, 564 
EosinopWia, 575 
Eosinophilic bronchitis, 10 
Epicritic elimination of nitrogen, 221 

polyuria, 181 
Epidemic cerebrospinal meningitis, 759 
Epiguanin, 252 
Episarkin, 252 
Epistaxis, Gull's renal, 373 
Epithelial casts, 377 

cells in feces, 119 

in gastric contents, 62 
in semen, 400 
in sputum, 1 1 
in urine, 367 
Erepsin, 102 
Erosive balanitis, 744 
Error in cell counting, 521 
Erythrasma, 172 
Erythroblasts, 545 
Erythrocytes, 541 

appearance of, 541 

color-index of, 465 

counting of, 509 

crenation of, 542 

degenerations of, 553 

formation of, 409 



Erythrocytes, functions of, 557 

isotonicity of, 555 

nucleation of, 545 

number of, 548 

pathological types of, 543, 553 

recognition of, in stains, ']2'] 

resistance of, 555 

rouleaux formation of, 542 

shape of, 543 

size of, 543 

staining properties of, 552 

structure of, 541 

variations of, 548 
Erythrocytometer, 511 
Erythrocytosis, 551 
Erythrodextrin, 36, 82 
Esbach's method for albumin, 287 
Essential albuminuria, 279 

pentosuria, 325 

renal hematuria, 373 
Esterification method of Fischer, 260 
Estivo-autumnal malaria, 624, 627 
Ethereal sulphates, 212, 214 
Euchlorhydna, 70 
EuglobuHn, 289 
European cat-fluke, 162 
European hook-worm, 154 
Eustrongylus gigas, 163 

visceralis, 163 
Ewald test-meal, 54 
Ewald and Siever's method for gastric 

motiHty, 85 
Exercise, effect of, on red cells, 549 

leucocytosis due to, 573 
Extraction method for fat, 773 
Extraneous material in sputum, 9 
Extruded intracellulars, 622 
Exudates, 736 

bacteriology of, 741 

chyloid, 739 

chylous, 739 

conjunctival, 47 

cytology of, 748 

formation of, 736 

hemorrhagic, 739 

obtaining of, "jy] 

peritoneal, 751 

pleural, 750 

properties of, 737 

purulent, 740 

putrid, 740 

serofibrinous, 739 

serous, 738 

urethral, 742 

False albuminuria, 275 
Famine fever, 631 
Fasciola hepatica, 160 

humana, 160 
Fasciolopsis buski, 161 
Fasting stomach, contents of, 60 
Fat in blood, 498 

in exudates, 739 

in feces, 115, 123 

'n milk, 772 

in urine, 364 
Fatty acids in blood, 498 

in exudates, 739 

in feces, 115, 123 



INDEX 



82s 



Fatty acids in sputum, 13 

in urine, 262 

casts, 374 

granules in leucocytes, 560 

stools, 107 
Favus, 168 
Febrile albuminuria, 278 

albumosuria, 293 

anemia, 595 

diseases, blood in, 606 

urine, 185 
Fecal vomitus, 61 
Feces, 99 

amount of, 104 

bacteriology of, 128 

bile acids in, 118 

biliary pigments in, 108, 117 

blood in, 108 

carbohydrates in, 116, 123 

chemical examination of, 121 

color of, 116 

concretions in, 118 

consistency of, 105 

crystals in, 120 

fat in, 114, 123 

food remnants in, 113 

formed, 105 

macroscopic examination of, 103 

marking of, loi 

microscopic examination of, 119 

morphological elements in, 119 

mucus in, 1 1 1 

normal, 99 

odor of, 106 

parasitology of, 133 

protein in, 114 

pus in, 113 

reaction of, 121 

tissue fragments in, 118 

total nitrogen of, 122 

total solids of, 121 

unformed, 105 
Fehling's test for glucose, qualitative, 

306 
Female secretions, 402 
Fermentation method of Schmidt, 124 

test for diphtheria bacillus, 40 
for glucose, 309, 320 
Fermentative dyspepsia, 117 
Ferments in blood, 509 

in feces, 102 

in gastric juice, 64, "]-! 

in leucocytes, 509 

in sputum, 6 

intestinal, 102 

in urine, 264 

pancreatic, 102 
Ferroc3^anide test for albumin, 285 
Ferrometer of Jolles, 508 
Fibers, elastic, 12, 115 

muscle, in feces, 115 
in gastric contents, 59 
Fibrin ferment, 440 

in blood, 443 

in urine, 297 

network, 443 

significance of, 443 

tests for, 297 
Fibrinogen, 440 



Fibrinous casts in sputum, 8 

in urine, 377 
Fibrinuria, 297 
Filaria Bancrofti, 634 

in blood, 634 

in urine, 364, 385 

nocturna, 634 

sanguinis hominis, 634 
Filariasis, 634 

Finely granular cells of Schultze, 560 
Fischer's esterification method, 260 

test-meal, 55 
Fish tape-worm, 146 
Fittipaldi's method for albumose, 293 
Fixation of complement, 680 

of smears, by chemicals, 530 
by heat, 528 
Fixed alkalinity, 191 
Flagella, staining of, 788 
Flagellata in feces, 140 

in sputum, 28 

in urine, 384 
Flat worms, 141 

Fleischl-Miescher hemometer, 456 
Flexner's serum, 759 
Florence's test for seminal fluid, 401 
Fluids, diluent for blood, 514, 520 
Fluke-worms, 147, 159 
Folin's method for acetone, 341 

for acidity of urine, 188 

for ammonia, 240 

for creatin, 258 

for creatinin, 256, 258 

for free mineral acidity, 190 

for indican, 273 

for sulphates, 214 

for urea, 232 

for uric acid, 246 

standard diet, 10 1 

Folin and Bell's direct nesslerization 

method for ammonia in urine,- 

241 

Folin and Denis' direct nesslerization 

method for total N in urine, 225 

method for lactose in milk, 773 
Folin and Macallum's method for am- 
monia in urine, 240 
Folin and McEllroy's test for sugar in 

urine, 305 
Folin and Peck's quantitative test for 

sugar in urine, 312 
Folin and Wright's simphfied Kje1dal;il 

method, 224 
Folin and Wu's method for creatin in. 
blood, 492 

c eatin in blood, 491 

non-protein N in blood, 470 

sugar in blood, 496 

urea in blood, 474 

uric acid in blood, 484 

uric acid in urine, 250 
Folin and Youngburg's direct nessleri- 
zation method for urea in urine, 
236 
Fontana's stain, 746 
Foreign bodies in sputum, 9 
Form of stools, 105 

Formaldehyd as preservative, 180, 775 
Formalin method for ammonia, 242 



826 



INDEX 



Formation of blood, 409 

of casts, 374 

of exudates, 736 
Fractional examination* of gastric juice, 

52 
Fourth venereal disease, 744 
Fraenkel's diplococcus, 25, 30, 651, 801 
Fragments of tissue in feces, 118 

in gastric contents, 63 

in sputum, 12 

in urine, 381 
Free hydrochloric acid, 65 

amount of, 69 

detection of, 66 

determination of , 67 

formation of, 51 

significance of, 70 

variations of, 70 
Freezing point of blood, 444 

of urine, 390 
Fresh blood, 525 
Friedlander's bacillus, 25, 46 
Frommer's test for .acetone, 339 
Fuchsin-aldehyd reaction for colon ba- 
cilli, 795 
Fucose, 325 
Functional albuminuria, 275 

diagnosis, 388 

hematuria, 373 
Functions of gastric ferments, 84 

of intestinal ferments, 102 

of leucocytes, 578 

of red cells, 557 
Fusaria mystax, 149 

vermicularis, 149 
Fusiform bacillus of Vincent, 40 
Futcher and Lazear's fixation method, 

531 
malarial stain, 539 

Gabbet's staining method, 21 
Gabritschewsky's polychromatophilia, 

552 
Gaffky's table, 23 
Galactosuria, 322, 329 
Gall stones in feces, 117 
appearance of, 117 
composition of, 117 
Gamete, 622 
Gametocyte, 622 
Gametoschizonts, 622 
Gangrenous balanitis, 744 
Gas bacillus, 133, 797 
Gases in blood, 508 
in feces, 125 
in gastric contents, 83 
Gastric carcinoma, 91 
contents, 50 
acetone in, 84 
after test meals, 62 
amino acids in, 83 
bacteria in, 62 
blood in, 59, 61, 83 
crystals in, 63 
digestion products in, 82 
epithelial cells in, 62 
food remnants in, 62 
fractional withdrawal of, 52 
from fasting stomach, 60 



Gastric carcinoma, contents from vom- 
it us, 60 

gases in, 83 

indirect examination of, 87 

macroscopic examination of, 57 

microscopic examination di, 62 

mucus in, 60, 62, 91 

obtaining of, 52 

protozoa in, 63 

pus in, 62 

tissue fragments in, 63 
crises, 89 
juice, 57 

acetic acid in, 64, 73, 77 

acidity of, 64 

amount of, 57 

butyric acid in, 64, 73, 77 

combined hydrochloric acid in, 

71 

composition of, 64 

deficit of hydrochloric acid in, 

73 

ferments of, 64, 78 
free hydrochloric acid in, 65, 92 
hyperacidity of, 65, 71, 88 
hypersecretion of, 89 
hypoacidity of, 65, 70 
in disease, 88 
lactic acid in, 74, 92 
organic acids in, 73 
Pawlow's work on, 51 
properties of, 57 
secretion of, 51 
motility, 84 
ulcer, 91 
Gastrin, 51 
Gastritis, acute, 90 
atrophic, 90 
chronic, 90 
Gastrosuccorrhea, 89 
Gelatin, nutrient, 782 
Genital organs, secretions of, 398 
Genito-urinary tuberculosis, 370, 383 
Gerhardt's test for diacetic acid, 342 
Ghoreyeb's stain for spirochaete, 745 
Giemsa's stain, 538, 745J 
Gigantoblasts, 547 
Gigantocytes, 544 
Glanders, bacillus of, 28 
Globular decolorization, 553 
Globulin-albumin ratio, 289 
Globulin in blood, 289, 468 
in exudates, 738 
in milk, 771 
in urine, 289 

significance of, 289 
tests for, 289 
Glomerular insufficiency, 396 
Glossina palpalis, 632 
Glucose agar, 783 

in the blood, 494 
in the urine, 298 

determination of, 312 
significance of, 298 
tests for, 303 
Glutoid capsules, 103 
Glycemia, 494 
Glycerin agar, 783 
Glycocholic acid, 117, 196, 349 



INDEX 



827 



Glycocoll, 260 

Glycogen in the blood, 497 

Glycosuria, 298 / 

alimentary, 299 

after poisoning, 302 

after use of drugs, 302 

diabetic, 300 

e saccharo, 299 

ex amylo, 299 

masked, 309 

neuro-hepatogenous, 301 

occult, 309 

physiologic, 298 

salt, 298 

transitory, 299 
Glycosuric acid, 351 
Glycuronic acid, 331 
Glycyl- tryptophan test, 93 
Gmelin's reaction for biliary pigments, 

348 
Goldhorn's stain, 638 
Goldschmiedt's test for glycuronic acid, 

333 
Gonococcus, 741, 805 
Gonorrhea, complement-fixation test in, 

702 
Gonorrheal conjunctivitis, 48 
stomatitis, 41 
threads, 378, 743 
urethritis, 743 
Goodman and Stern's method for albu- 
min, 288 
Gout, blood in, 605 

perinuclear granules in, 560 
urine in, 245 
Gowers' hemoglobinometer, 455 
Gram-negative organisms, 742 
Gram-positive organisms, 742 
Gram's stain, 742 
Granular casts, 375 

cells in blood, 560, 562, 564 
in prostatic fluid, 399 
in sputum, 10, 11 
degeneration, 552 
Granules in blood, acidophile, 562 
basophile, 553, 562 
of Grawitz, 553 
Ehrlich's a, 562 
/3, 562 
% 562 
5.563 
e, 560 
eosinophile, 562 
fatty, 565 
glycogen, 565 
Grawitz, 553 
hemoconien, 581 
in malaria, 622, 623, 625 
mast cell, 562 
melanin, 622, 623, 625 
Neusser's, 560 
neutrophile, 560 
oxyphilic, 562 
perinuclear, 560 
sudanophile, 565 
in sputum, 1 1 
Grape-sugar in urine, 298 
Gravel in urine, 385 
Grawitz' basophilia, 553 



Green sputum, 3 

vomitus, 61 
Griess-Ilosvay reagent, 36 
Grinders' rot, 4 
Gross' method for trypsin, 102 
Ground itch, 155 
Grouping of blood, 724 
Gruber-Widal reaction, 670 
Guaiac test for blood, 109, no, 728 
Guanin, 252 

Gull's renal-epistaxis, 373 
Gum, animal, in urine, 331 
Gummatous lymphoma, 603 
Gunning's mixture, 222 

test for acetone, 338 
Giinzburg's package, 87 

reagent, 66 

test for free hydrochloric acid, 66 
Gynecophorus haematobius, 385, 646 

Haeser's coefficient, 194 
Haines' coefficient, 194 

test for glucose, qualitative, 306 
quantitative, 316 
Haldane and Smith's method for volume 

of blood, 411 
Halitus sanguinis, 418 
Hammarsten's test for biliary pigments, 

348 
Hammerschlag's method for specific 

gravity, 438 
for pepsin, 79 
Haptines, 656 
Haptophore, 656 
Hard chancre, organisms of, 744 
Hart's method for /^-oxybutyric acid, 

343 
Harvest bug, 165 
Hayem's solution, 514 
Hay fever, 45 

Hay's test for bile acids, 349 
Head louse, 166 
Heart disease cells, i, 32 

pleurisy of, 751 
Heat fixation of smears, 528 
Heat test for albumin, 281 
Hehner-Maly method for organic acids, 

72> 
Heller's table for examination of calculi, 

387 

test for albumin, 282 
for hemoglobin, 296 
Hemameba malariae, 623 

vivax, 620 
Hemamebiasis, 619 
Hematemesis, 3, 61 
Hematin, 452 

hydrochlorate, 453, 731 
Hematoblasts, 580 
Hematochyluria, 636 
Hematocrit, 413 
Hematogenous albuminuria, 279 

albumosuria, 294 

urobilinuria, 269 
Hematoglobulin, 450 
Hematoidin in the blood, 453 

in sputum, 11, 13 

in urine, 346 
Hematopoietic organs, 409, 582 



828 



INDEX 



Hematoporphyrin in blood, 453 

in feces, 108 

in stains, 732 

in urine, 346 
Hematoporphyrinuria, 346 
Hematuria, 372 

angioneurotic, 373 

constitutional, 373 

essential, 373 

extra-renal, 373 

functional, 373 

idiopathic, 373 

renal, 373 
Hemin, 453, 731 
Hemochromogen, 451 
Hemoconien, 581 
Hemocytometer of Durham, 522 

of Oliver, 523 

of Thoma-Zeiss, 510 
Hemoglobin in blood, 448 
amount of, 449 
derivatives of, 450 
estimation of, 454 
properties of, 448 
variations of, 464 

in sputum, 3, 5, 11, 13 

in urine, 295, 346 
tests for, 296 

quotient, 465 

value, 465 
Hemoglobinemia, 295 
Hemoglobinemic degeneration, 553 
Hemoglobinometer of Dare, 459 

of Oliver, 461 

of Tallqvist, 463 
Hemoglobinuria, 295, 346 

paroxysmal, 296 

significance of, 295 

tests for, 296 
Hemolysins, 658 
Hemolysis, 658 
Hemolytic anemia, 594 
Hemometer of Fleischl-Miescher, 456 

of Sahli, 460 
Hemophilia, renal, 373 
Hemoptysis, 3 
Hemo-renal index, 477 
Hemorrhage, anemia due to, 592 

occult, 108 
Hemorrhagic exudate, 739 

nephritis, 373 
Hemosiderin, II, 454 
Hepatic insufficiency, 125, 229, 301 
Hepatogenous albumosuria, 294 

urobilinuria, 269 
Herman-Perutz reaction, 717 
Herpes tonsurans, 169 
Heteroalbumosuria, 290 
Heterochylia, 89 
Heteroxanthin, 252 
Hexamitus duodenalis, 140 
Hexone bases, 83, 91, 260 
High ratio organisms, 794 
Hippuric acid in urine, 261, 363 
Hirschfeld and Klinger's test, 719 
Histidin, 260 

Histoplasma capsulatum, 634 
Histoplasmosis, 634 
Hodgkin's disease, 601 



Hoffman's bacillus, 40, 790 
Homogentisic acid, 351 
Hopkin's method for uric acid, 246 
Hoppe-Seyler's colorimetric pipet, 455 
Howell's immature nucleated reds, 546 

mature nucleated reds, 546 
Huppert-Messinger method for acetone, 

339 

Hyaline casts, 374 
Hydatid cysts, 146, 754 
Hydatidiform degeneration, 406 
Hydremia, 412 
Hydrobilirubin in stools, 107 
Hydrocele agar, 784 

fluid, 753 
Hydrochloric acid in gastric juice, 51 

amount of, 69 

combined, 71 

deficit, 73 

estimation of, 67 

free, 65 

physiologically active, 72 

tests for, 66 
Hydrogen ion concentration, 419, 428 

of blood, 419, 428 

of culture media, 781 

of gastric juice, 65 

of saliva, 35 

of spinal fluid, 756 

of urine, 188 
Hydrogen sulphide in gastric contents, 
83 

in urine, 213 
Hydronephrosis, 754 
Hydrops folliculorum Graafii, 752 
Hydroquinone-acetic acid, 351 
Hydruria, 181 
Hymenolepis diminuta, 145 

flavopunctata, 145 

murina, 144 

nana, 144 
Hypalbuminosis, 467 
Hyperacidity of gastric juice, 65, 71 
Hyperalbuminosis, 467 
Hyper chlorhydria, 71, 88 
Hyperglycemia, 494 
Hyperinosis, 443 
Hypermotility of stomach, 85 
Hypersecretion of gastric juice, 80 
Hypertonic solutions, 555 
Hyphogenous sycosis, 169 
Hypinosis, 443 
Hypochlorhydria, 70 
Hypostatic albuminuria, 278 
Hypotonic solutions, 555 
Hypoxanthin, 240, 252 

Ice-box fixation, 691 

Idiopathic enterogenous cyanosis, 451 

pentosuria, 325 
Ilosvay's reagent, 36 
Immature nucleated reds of Howell, 546 
Immunity, 660 
Inactivation of serum, 658 
Inanition, anemia due to, 593 
Incubation, 784 
Index, bacillary, 661 

color, 465 

hemoglobin, 465 



INDEX 



829 



Index, McLean's, of urea excretion, 478 

of acid excretion, 189 

of urea excretion, 478 

opsonic, 661 

phagocytic, 661 

volume, 414 
India ink method for spirochsete, 745 
Indican, 270 
Indicaniuria, 270 
Indigo blue in urine, 270 

red in urine, 272 
Indirect examination of gastric contents, 

87 
Indol-acetic acid, 274 
Indoxyl-potassium sulphate in urine, 270 
Infectious diseases, blood in, 606 

jaundice, 643 
Influenza, bacillus of, 26, 791 

sputum in, 33 
Infusoria, in feces, 141 

in sputum, 28 

in urine, 384 
Inorganic constituents of blood, 503 

of urine, 197 
Inoscopy, 741 
Inosite in urine, 331 
Insecta, 166 
Insufficiency, digestive, 113 

glomerular, 396 

hepatic, 125, 229, 301, 322 

motor, 84 

renal, 396 

tubular, 396 
Intermittent albuminuria, 277 
Intestinal concretions, 118 

digestion, 102 

juices, 102 

obstruction, 114 

parasites, 133 

sand, 118 
Iodide of potassium test 

of absorptive power, 86 

of renal function, 389 
Iodoform test for acetone, 338 

for lactic acid, 76 
lodophilia, 565 
Iron in the blood, 507 

in urine, 218 
Irritation forms of leucocytes, 565 
Isoagglutinins, 723 
Isohemolysins, 723 
Isomaltose in urine, 331 
Isotonicity of red cells, 555 
Isotonic solutions, 555 
Itch parasite, 165 

Jaeger and Heubner's diplococcus, 759 
Jaffe's test for creatinin, 256 

for indican, 271 
von Jaksch's anemia, 590 
Jansky's blood groups, 724 
Japanese liver-fluke, 162 
Jaundice, blood in, 643 

sputum in, 4 

urine in, 185, 347 
Jecorin, 493 
Jenner's stain, 536 
Jigger, 168 
Jolles' ferrometer, 508 



Jousset's fluid, 741 
Juice, gastric, 57 

intestinal, loi 
Justus' test for syphilitic blood, 616 

Kahler's disease, 291 
Kala-azar, blood in, 634 

parasite of, 634 
Karyomorphism of neutrophiles, 561 
Kastle and Loevenhart's method for 

lipase, 265 
Kathrein's test for bile pigments, 347 
Kelling's test for lactic acid, 75 
Kendall and Day's method for typhoid 

bacilli, 132 
Kendall and Ryan's double sugar med- 
ium, 132 
Ketosis, 335 
Kidney, abscess of, 370 

acute inflammation of, urine in, 
183, 185, 194, 199, 206, 222, 
279 
amyloid disease of, urine in, 183, 

186,266,280,289 
cancer of, 372, 381 
chronic inflammation of, urine in, 
178, 183, 185, 194, 199, 206, 
279 
echinococcus cysts of, 384 
hemorrhagic lesions of, 372 
hydronephrotic cysts of, 754 
malignant disease of, 372, 381 
stones, 385 

suppurative lesions of, 370 
syphilitic disease of, 279 
tubercular, 370 
Kjeldahl's method for nitrogen, 222 
Klebs-Loffler bacillus, 38, 789 
Knop-Hiifner method for urea, 230 
Koch's bacillus, 18, 132, 389, 654, 741 
comma bacillus, 61, 129, 799 
tuberculin, 18, 663 
Koch- Weeks bacillus, 48 
Kohlrausch's method for electric con- 
ductivity, 390, 445 
Krabbea grandis, 147 
Kreatin (see Creatin), 253, 258, 492 
Kreatinin (see Creatinin), 254, 256, 490 

Lab, 64, 81 

Labor, albuminuria following, 276 

Lactic acid in blood, 498 

in gastric contents, 74 
in carcinoma, 92 y 

significance of, 74 
tests for, 75 

in urine, 262 
Lactose in milk, 773 

in urine, 329 

test for renal function, 389 
Lactosuria, 329 
Lactose-litmus agar, 783 
Laiose, 325 
Laking of blood, 555 
Lamblia intestinalis, 140 
Landau's test, 717 
Lange's colloidal-gold test, 763 
Large lymphocytes, 558 

mononuclear leucocytes, 559 



830 



INDEX 



Larvae in aural secretions, 47 

in feces, 151, 152, 156 
Laveran's malarial organism, 619 
Layers of sputum, 5 
Lead, anemia due to, 595 

basophilia in poisoning, 553 
Lecithin globules in semen, 399 
Legal's test for acetone, 338 
Leiner's test for casein, 115 
Leishman-Donovan bodies, 634 
Le Nobel's test for acetone, 338 
Leone's test for albumin, 286 
Leo's method for chymosin, 81 
Leprosy, bacillus of, in sputum, 24 

blood in, 616 
Leptodera intestinalis et stercoralis, 150 
Leptospira icterohemorrhagica, 643 

icteroides, 641 
Leptothrix buccalis, 38 

in sputum, 15 
Leptus autumnalis, 165 
Leube's test of gastric motility, 85 
Leucin in sputum, 13 

in urine, 259, 361 
Leucocytes, 557 

appearance of, 557 

basophilic, 562 

counting of, 520 

degenerated forms of, 565 

differential counting of, 566 

eosinophilic, 562 

ferments of, 509 

formation of, 410 

functions of, 578 

granules in, 560, 562, 564 

in blood, 557 

in exudates, 748 

in feces, 120 

in gastric contents, 62 

in milk, 777 

in sputum, 10 

in urine, 369 

irritation forms, 565 

karymorphism of, 561 

large mononuclear, 559 

lymphocytes, 558 

mast-cell, 562 

myelocytes, 563 

neutrophilic, 564 

number of, 567 

oxyphilic, 562 

pigmented, 563 

polymorphonuclear, 560 j 

small mononuclear, 558 

splenocytes, 559 

transition forms of, 565 

types of, 558 

variations in number of, 567, 577 
Leucocytic crystals in sputum, 14 
Leucocytometer, 512 
Leucocytosis, 568 

agonal, 572 

antemortem, 572 

cachectic, 572 

eosinophilic, 575 

infectious, 571 

inflammatory, 57] i 

mast-cell, 577 

mixed, 574 



Leucocytosis of digestion, 569 

of pregnancy, 570 

of the new-born, 570 

polymorphonuclear, 568 

post-hemorrhagic, 572 

therapeutic, 573 
Leucohydrobilirubin in feces, 107 
Leucopenia, 577 
Leucorrhea, 403 
Leucourobilin, 107 
Leukanemia, 590 
Leukemia, 596 

acute, 600 

lymphatic, 599 

mixed, 596 

splenomyelogenous, 596 
Levaditi and Manouelian's method for 

staining treponema, 746 
Levulose in urine, 322 

determination of, 323 

recognition of, 323 

significance of, ;^22 

tests for, 323 
Levulosuria, 322 

Levy, Rowntree and Marriott's test for 
ion concentration of blood, 433 
Lieben's test for acetone, 338 
Lientery, 113 

Limitations of blood examinations, 732 
Limnaea truncatula, 161 
Lipacidemia, 498 
Lipaciduria, 262 
Lipase in gastric juice, 64, 82 

in pancreatic juice, 102 

in urine, 265 
Lipemia, 498, 604 

Lipliawsky's test for diacetic acid, 343 
Lipuria, 364 
Liquor sanguinis, 416 
Lithemit; diathesis, 245 
Litmus-glucose agar, 783 

lactose agar, 783 

milk, 784 
Liver, abscess of, sputum in, 33 

insufficiency of, 125, 229, 301, 322 
Lobar pneumonia, blood in, 607 

chlorids in urine of, 199 

organism of, 25, 31 

sputum in, 31 
Lochia alba, 405 

cruenta, 405 

rubra, 405 

serosa, 405 
Lofffer's antiformin method, 18 

methylene blue, 21 
Lohnstein's saccharometer, 320 
Long's coefficient, 194 
Lordotic albuminuria, 2"]"] 
Low ratio organisms, 794 
Luetin reaction, 665 
Lumbar puncture, 755 
Lung, abscess of, sputum in, 33 

fluke in sputum, 29 

inflammation of, 31, I99» 607 

stones, 8 
Lymphatic leukemia, 599 

pseudoleukemia, 601 
Lymphemia, 599 
Lymphocytes, 558 



INDEX 



831 



Lymphocytosis, 574 
Lymphopenia, 574 
Lymphosarcoma, 602 
Lysins, 658 
Lytic action, 658 

Macrocytes, 544 
Macrocythemia, 544 
Macrocytosis, 544, 588 
Macrogamete, 622 

Magnesium ammonium phosphate in 
feces, 120 

in sputum, 13 

in urine, 366 

phosphate in urine, 203, 366 

salts in urine, 216 

soaps in feces, 116 
Malaria, blood in, 630 

fresh blood in, 620 

mosquito theory of, 619 

parasites of, 620, 623, 624 
estivo-autumnal, 623, 627 
quartan, 623, 627 
tertian, 620, 626 

stained smears in, 625 
Malarial pigment, 454 
Male secretions, 398 
Malfatti's method for ammonia, 242 
Malignant disease, blood in, 616 
gastric juice in, 91 
urine in, 372, 381 

lymphoma, 602 

pleurisy, 751 
Mallory's stain for Negri bodies, 176 
Malone's test for pregnancy, 260 
Maltose in urine, 330 
Mammary secretions, 766 
Maragliano's endoglobular degeneration, 

553 
Marechalt's test for bile pigment, 347 
Marriott's method for alkali reserve of 

blood, 436 
Marshall's method for urea, 234 
Martius and Luttke's method for HCl, 

71 

Marx's fluid, 728 
Mast-cell granules, 562 

leucocytosis, 577 
Masturbators, albuminuria of, 277 
Mature nucleated reds of Howell, 546 
May-Grunwald stain, 537 
McCrudden's method for calcium, 217 
McLean's Index of urea excretion, 478 
Meals, test, 54 
Measles, blood in, 611 
Medicinal leucocytosis, 573 
Medico-legal aspects of blood, 727 

of semen, 401 
Megaloblasts, 546 
Megalocytes, 544 
Megalogastria, 85 
Megastoma entericum, 140 

intestinale, 140 
Melanin, 186, 350, 454 
Melanogen, 186 
Melanuria, 350 
Membranous dysmenorrhea, 405 

enteritis, 112 

ureteritis, 266 



Meningeal fluid, examination of, 754 
Meningitis, epidemic cerebrospinal, 759 

tubercular, 760 
Meningococcus of Bonome, 759 

of Weichselbaum, 759, 806 
Menstruation, 405 
Messinger method for acetone, 339 
Metalbumin in ovarian cysts, 752 
Metamyelocytes, 561 
Methemoglobin, 450 
Methylene azure stains, 537 

blue in urine, 186, 353 
stains, 21, 533 
Methylphenylosazon, 323 
Methyl red test, 795 
Methylxanthin, 253 
Mett's method for pepsin, 79 
Meyer's test for blood, 729 
Microblasts, 546 

Micrococcus catarrhalis in sputum, 17, 
807 

tetragenus in sputum, 17 
Microcytes, 543 
Microgametes, 622 
Microgametocytes, 622 
Microscopy of blood, 524 

of exudates, 748 

of feces, 119 

of gastric contents, 62 

of milk, 775 

of semen, 398 

of sputum, 9 

of urine, 354 
Microsporon Audouini, 170 

furfur, 170 

minutissimum, 172 

scorteum, 169 
Miescher's hemoglobinometer, 456 
Milk, 766 

appearance of, 768 

ash of, 769 

bacteriology of, 775 

coagulation of, 769 

compositon of, 767 

cow's, 768 

curds in stools, 115 

fat of, 772 

human, 767 

lactose of, 773 

microscopy of, 775 

preservatives in, 774 

properties of, 767 

protein of, 770 

reaction of, 769 

specific gravity of, 768 

sugar of, 773 

total solids, 769 
Milk-curdling ferment, 64, 81 
Milky zone, 621 
Mineral acidity of urine, 190 
Minot's method for grouping blood, 726 
Mintz's method for free HCl, 68 
Mitochondria, 545 
Mixed infection in tuberculosis, 23 

leucocytosis, 574 
Molds in aural secretions, 47 

in buccal secretions, 36 

in sputum, 16 
Moller's method for spores, 788' 



832 



INDEX 



Monocalcium phosphate in urine, 364 
Monocercomonas hominis, 140 
MonochromatophiHa, 552 
Mononuclears, basophile, 564 

eosinophile, 564 

large, 559 

neutrophile, 564 

small, 558 
Moore and Wilson's test for alkalinity, 

418 
Morax-Axenfeld diplobacillus, 48 
Morner-Sjoqvist method for urea, 233 
Morner's mucin-like bodies in urine, 266 

test for tyrosin, 363 
Morning sputum, i 
Moro's tuberculin reaction, 663 
Morphology of blood, 524 

of blood-forming organs, 582 
Mosquito anopheles, 619 

culex, 641 

cycle of malarial parasites, 629 

stegomyia, 641 

theory of malaria, 619 
of yellow fever, 641 
Moss' blood groups, 724 

method for determining, 726 
Motility of intestine, 104 

of stomach, 84 
detection of, 85 
types of, 84 
Motor insufficiency, 84 
Moults, 164 

Mouth, inflammation of, 40 
catarrhal, 40 
gonorrheal, 41 
mycotic, 41 
ulcerative, 40 
ulceromembranous, 40 

secretions of, 35 
Much's method for tubercle bacilli, 22 . 
Mucin in ovarian cysts, 752 

in sputum, 3 

in urine, 265 
Mucinophiles, 563 
Mucoid material in urine, 367 

sputum, 5 
Mucopurulent sputum, 5 
Mucor in sputum, 16 
Mucous corpuscles, 367 

threads in urine, 371, 743 
Mucus in feces, 1 1 1 

appearance of, 1 1 1 
detection of, 1 1 1 
significance of, 112 

in gastric contents, 60, 62, 91 

in sputum, 5 

in urine, 265 
Mullern's blood stain, 533 
Murexid test, 357 
Muscle fibers in feces, 114 

in gastric contents, 60 

in sputum, 12 
Mycelial casts in sputum, 8 
Myelemia, 596 

Myelin granules in sputum, 1 1 
Myeloblasts, 559 
Myelocytes, 563 

basophile, 564 

eosinophije, 564 



Myelocytes, neutrophile, 564 

Cornil's, 564 

Ehrlich's, 564 
Myelocytosis, 574 
Myelogenous leukemia, 596 
Myeloid leukemia, 596 
Myelopathic albumosuria, 291 
Myxedema, blood in, 606 
Myxococcidium stegomyiae, 641 
Myxoid cyst of ovary, 752 
Myxoma of the placenta, 406 

Nakayama's test for bile pigments, 348 
Naphthoresorcin test, 333 
Nasal secretion, 44 

bacteria in, 44 

composition of, 44 

concretions in, 46 

pathology of, 44 

spinal fluid in, 45 
Negri bodies, 175 
Neisser's diplococcus, 741, 805 

stain for diphtheria bacillus, 39 
Nematodes in feces, 148 

in urine, 385 
Nephritic albuminuria, 279 

hematuria, 372 

oliguria, 183 

pleurisy, 751 
Nephritis, acute, 183, 185, 194, 199, 206, 
222, 280 

albuminuria of, 279 

chronic diffuse, 194, 199, 206, 280 
interstitial, 183, 187, 280 
parenchymatous, 183, 185, 280 

hemorrhagic, 372 

suppurative, 370 

syphilitic, 279 

unilateral, 396 
Nervous dyspepsia, 90^ 

type of albuminuria, 279 j] 
of polyuria, 182 
Neubauer and Fischer's test for gastric 

carcinoma, 93 
Neuberg and Wohlgemuth's method for 

pentose, 327 
Neuberg's test for glycuronic acid, 334 

for levulose, 323 
Neusser's granules, 560 
Neutral calcium phosphate in urine, 364 

dyes, 532 

stains, 532 

sulphur in urine, 213 
Neutrophile cells, 560, 564 

granules, 560 
Neutrophilic karyolobism, 561 
New-born, albuminuria of, 276 

leucocytosis of, 570 
Night urine, 181 
Nikiforoff's. method of fixation of smears, 

530 
Nitric acid test for albumm, 282 
Nitrites in saliva, 36 
Nitrogen of sputum, 2 

of urine, 218 

partition of blood, 469 
of urine, 219 
Nitrogei^ous balance, 218 

bodies in blood, 469 



INDEX 



833 



Nitrogenous bodies in exudates, 738 
in feces, 122 
in gastric contents, 93 
in milk, 770 
in sputum, 2, 5 
in transudates, 737, 
in urine, 218 
allantoin, 261 

alloxyproteic acid, 213, 261 
amino-acids, 259 
ammonia, 237, 487 
creatinin, 253, 490 
hippuric acid, 261, 363 
oxyproteic acid, 261 
purin bases, 252 
total, 218, 469 
undetermined, 259 
urea, 228, 474 
uric acid, 242, 481 
equilibrium, 218 
Nitroprussid test for acetone, 338 
Nocht's malarial stain, 539 
Noguchi's antigen, 685 
butyric acid test, 762 
luetin reaction, 665 
method for spirochete, 639, 744 
modification of Wassermann test, 
692 
Nondiffusible alkalinity of blood, 418 
Nonne's test of cerebrospinal fluid, 762 
Nonprotein nitrogen of blood, 469 
Normal feces, 99 

salt solution, 555 
Normoblasts, 545 
Normocytes, 543 
N^Dse, secretions of, 44 
Nubecula, 184 
Nubecular threads, 184, 378 
Nucleated red cells, 545 

Howell's immature, 546 
mature, 546 
Nuclein bases, 122, 196, 252, 359 
Nucleinic acid, 245, 266 
Nucleo-albumin in urine, 266 
Number of blood plates, 580 
of leucocytes, 567 
of red cells, 548 
of stools, 104 

of tubercle bacilli in sputum, 22 
Nummular sputum, 5, 6 
Nutrient agar, 782 
broth, 781 
gelatin, 782 
Nutrition, effect of, on blood, 549, 

■ 593 

Nycturia, 181 

Nylander's test for glucose, 308 

Obermayer's test for indican, 271 
Obermeier's spirillum, 631 
Obtaining blood, 415, 686 

exudates, 737 

gastric contents, 52 
Occult blood in feces, 108 
Ochronosis, 351 
Octomitus hominis, 140 
Odor of blood, 418 

of exudates, 738 

of feces, 106^ 



Odor of gastric contents, 59 

of sputum, 4 

of urine, 187 
Oidium albicans, 16, 41 
Oil test-breakfast, 102 
Oligemia, 418 
Oligochromemia, 465 
Oligocythemia, 550 
Oliguria, 182 
Oliver's hemocytometer, 524 

hemoglobinometer, 461 

test for bile acids, 349 
Oocyst, 629 
Ookinet, 629 

Operation, blood after, 603 
Opisthorchis felineus, 162 

sinensis, 162 
Oppler-Boas bacillus, 62 
Opsonic index, 661 
Opsonins, 660 
Optical activity of carbohydrates, 317 

of conjugated glycuronates, 318 

of glycuronic acid, 318 

of urine, 195 
Oral secretions, 35 
Orcein stain, 12 
Orcin test for pentose, 327 
Organic acidity of urine, 190 

acids in gastric contents, 73 
in urine, 262 
Organized sediments in urine, 367 

bacteria, 381 

blood cells, 372 

casts, 374 

epithelial cells, 367 

mucoid material, 367 

parasites, 384 

pus cells, 369 

spermatozoa, 381 

tissue fragments, 381 
Origin of casts, 374 

of leucocytes, 410 

of red cells, 409 
Orthostatic albuminuria, 277 
Orthotic albuminuria, 277 
Osier's disease, 551 
Osmotic pressure of blood, 443 

of urine, 390 
Otomycosis, 47 
Ova in feces, 133, 157 

in sputum, 29 

in urine, 385 

of anopheles, 619 

of intestinal parasites, 157 
Ovarian cysts, 752 

colloid, 752 

dermoid, 753 

myxoid, 752 

papillary, 753 

serous, 752 
O voids in malarial blood, 625 
Oxalate of calcium in sputum, 14 

in urine, 263, 359 

calculi, 386 
Oxalic acid in urine, 263 

amount of, 263 

determination of, 264 

origin of, 263 

variations of, 263 



834 



INDEX 



Oxaluria, 263 

Oxaluric acid, 263 

Oxid of bismuth in feces, 107 

/3-oxybutyric acid in urine, 343 

determination of, 344 

significance of, 343 

tests for, 343 
Oxyhemoglobin, 450 

p-oxyphenyl-a-amino-propionic acid, 362 
OxyphiHc cells, 562 

granules^ 562 
Oxyproteic acid, 196, 261 
Oxyuris vermicularis, 149 
Ozena, 46 

Palpation, albuminuria due to, 277 
Paludism, 619 
Pancreatic cysts, 754 

disease, feces in, 114 

fluid, 754 

juice, 102 

composition of, 102 
ferments of, 102 
insufficiency of, 114 
Panoptic staining, 532, 536 
Papillary cysts of the ovary, 753 
Pappenheim's amblyochromatic erythro- 
blasts, 546 

heteroplastic promyelocytes, 564 

method for tubercle bacillus, 21 . 

stain for blood smears, 536, 539 

trachyochromatic erythroblasts, 545 
Paracresol, 350 
Paragonimus westermanii, 29 
Paramecium coli, 141 
Paramucin, 753 
Parasites, anemia due to, 594 

eosinophilia due to, 576 

in blood, 619 

in feces, 133 

in sputum, 28 

intestinal, 133 

in urine, 384 

malarial, 619 

of the skin, 164 
Parasitology of the blood, 619 

of the feces, 133 

of the skin, 164 
Paratyphoid bacillus, 131, 649 
Paraxanthin, 252 

Parenchymatous nephritis, acute, 183, 
185, 194, 199, 206, 222, 280 

chronic, 183, 184, 280 
Parhemoglobin, 450 
Parovarian cysts, 753 
Paroxysmal hemoglobinuria, 296 

polyuria, 181 
Pathogenic bacteria in blood, 646 

in exudates, 741 

in feces, 128 

in gastric contents, 62 

in milk, 775 

in sputum, 17 

in urine, 381 
Pea-soup stools, 130 
Pediculus capitis, 166 

pubis, 166 

vestimenti, 166 
Penicillium glaucum, 16 



Pentose in urine, 325 

determination of, 327 

significance of, 325 

tests for, 326 
Pentosuria, alimentary, 325 

essential, 325 

idiopathic, 325 

intrinsic, 325 
Penzoldt and Faber's test, 86 
Pepsin in gastric juice, 64, ^^ 
activity of, 77 
detection of, 78 
determination of, 79 
significance of, 78 

in urine, 265 
Pepsinogen, 'j'] 
Peptic glands, 50 
Peptone in the blood, 468 

in gastric contents, 82 

in urine, 295 
Peptonuria, 295 
Perez' bacillus, 46 

Perforating empyema, sputum in, 34 
Pericardial fluid, 750 
Perinuclear granules of Neusser, 560 
Periodic albuminuria, 277 

polyuria, 181 
Peritoneal exudates, 751 

composition of, 751 

cytology of, 751 
Permeability of red cells, 555 

renal, 390 
Pernicious anemia, 587 
Pertussis, blood in, 613 

organism of, 26, 791 
Pessary forms of red cells, 541 
Petroff's method for tubercle bacilli, 19 
Pettenkofer's test, 350 
Petzetaki's test for tuberculosis, 354 
Pfeiffer's bacillus, 26, 33, 791 
Phagocytic cells, 4, 660 

index, 661 
Phagocytosis, 660 
Pharyngomycosis leptothrica, 38 
Phenol in feces, 99 

in urine, 350 
Phenolphthalin test, 729 
Phenolsulphuric acid, 350 
Phenolsulphonephthalein test, 394 
Phenoltetrachlorphthalein test, 125 
Phenylglucosazon, 310 
Phenylhydrazine test for glucose, 310 
Phloridzin test, 394 
Phloroglucin test for pentose, 326 

vanillin test for HCl, 66 
Phosphates, calcium, 203, 216 

in blood, 507 

in sputum, 13 

in urine, 203 

magnesium, 203, 218, 366 

magnesium-ammonium, 13, 120, 366 

triple, 13, 120, 366 
Phosphatic calculi, 388 

diabetes, 206 

sediments in urine, 364, 365 
Phosphaturia, 203, 365 
Phosphorus containing proteins, 266 

poisoning, blood in, 595 
urine in, 229 



INDEX 



83s 



Phthirius inguinalis, 166 
Phthisis, blood in, 615 

hemoptysis in, 3 

melanotica, 11 

sputum in, 30 

stone-cutters', 4 
Physiological albuminuria, 275 

glycosuria, 298 

salt solution, 555 

variations in blood cells, 548, 569 
Physiologically active HCl, 72 
Physis intestinalis, 156 
Pigment, bile, in blood, 502 
in feces, 108, 117 
in gastric contents, 59, 61 
in sputum, 3 
in urine, 346 

blood, in feces, 108 

in gastric contents, 59, 61, 83 
in s,outum, 3, 5, 11, 12, 14 
in urine, 346 

coal, in sputum, 4, 34 

in leucocytes, 4, 621, 623, 624 

in red Cells, 552, 620 

of blood, 448 

of urine, 267, 346 
Pin worm, 149 
Pineapple test, 77 
Piroplasma hominis, 634 
Piroplasmosis, 634 

von Pirquet's tuberculin reaction, 664 
Placenta cells, 406 
Plague bacillus, 27 
Plasma, 413 

chlorids, 503 
Plasmodium falciparum, 624 
quotidianum, 623 

malarias, 623 

precox, 624 

tenue, 620 

vivax, 620 

variety minuta, 620 
Platelets, blood, 580 
Platodes in feces, 141 
Plehn's karyochromatophilic granules, 

626 
Plethora, cellular, 412 

serous, 412 

true, 412 

vera, 412 
Pleuritic effusions, 750 

cytology of, 750 

withdrawal of, 737 
Plugs, Dittrich's, 6 

prostatic, 400 
Pneumococcus of Fraenkel, 25, 31, 651, 
801 

pleurisy, 750 

types of, 26 
Pneumoliths, 8 
Pneumonia, blood in, 607 

chlorids in urine in, 198 

organism of, 25, 31, 651, 801 

sputum in, 31 

urine in, 198 
Pneumonoconioses, 34 
Pneumonomycosis aspergillina, 16 
Poikilocytes, 544 
Poikilocytosis, 544 



Poisons, blood, 595 
Polariscope, 317 

Polariscopic method for glucose, 317 
Poliomyelitis, acute anterior, 760 
Polychromasia, 552 

Polychromatophilia of Gabritschewsky, 
552 

of Maragliano, 553 
Polychrome dyes, 536 
Polychromemia, 551 
Polycythemia, 551 
Polyglobulia, 551 
Polymorphonuclear basophiles, 562 

eosinophiles, 562 

neutrophiles, 560 
Polymorphonuclear neutrophiliosis, 568 
Polynucleosis, 568, 749 
Polyplasmia, 412 
Polyuria, 181 

epicritic, 182 

paroxysmal, 182 

periodic, 182 
Poor, anemia of the, 593 
Pork tape-worm, 143 
Posterior urethritis, 371 
Post hemorrhagic anemia, 592 

infectious albuminuria, 278 
Postural albuminuria, 277 
Potassium acid urate sediment, 358 

ferrocyanide test for albumin, 285 

iodide test, 86 

for renal function, 389 

of blood, 447 

of urine, 216 

sulphocyanate in saliva, 35 
Precipitinophore, 660 
Precipitins, 660 
Precipitin test for blood, 677 
Pregnancy, albuminuria of, 276 

ammonia in urine of, 238 

anemia of, 593 

blood in, 570 

leucocytosis of, 570 

sero-diagnosis of, 703 

urine test in, 260 
Preparation of blood smears, 526 

culture media, 780 

vaccines, 809 
Preservation of urine, 179 
Primary anemia, 585 

pernicious anemia, 587 

proteoses, 290 

tubercular pleurisy, 750 
Products of gastric digestion, 82 

of intestinal digestion, 102 
Progressive pernicious anemia, 587 
Promyelocytes, 564 
Propepsin, 51, 78 
Prostatic casts, 400 

fluid, 398 

plugs, 400 

secretion, 398 
Prostatitis, 371 
Prostatorrhea, 400 
Protalbumose, 290 
Protein in blood, 466 

in exudates, 738 

in feces, 114 

in gastric contents, 82, 93 



836 



INDEX 



Protein in milk, 770 

in sputum, 5 

in urine, 274 
quotient of serum, 468 
Proteoses in urine, 290 
Prothrombase, 440 
Protoryxomyces coprinarius, 140 
Protozoa in blood, 619 

in feces, 134 

in gastric contents, 63 

in sputum, 28 

in urine, 385 
Provocative Wassermann test, 701 
Prowazek-Greeff trachoma bodies, 48 
Prune- juice sputum, 3, 31 
Pseudo casts, 379 

diphtheria bacillus, 40, 790 

elastic tissue, 12 

gall stones, 118 

globulin, 289 

hemoglobin, 450 

leukemia, 601 
infantum, 590 

mucin, 753 

nucleation, 553 

parasites, 156 

rhabditis stercoralis, 150 
Ptomaines in feces, 123 

in urine, 360 
Ptyalin, 35, 36, 82 
Ptyalism, 37 

Puberty, albuminuria of, 278 
Puerperal infection, 404 
Pulex irritans, 168 

penetrans, 168 
Pulmonary actinomycosis, 2'] 

gangrene, 33 

hemorrhage, 3 

tuberculosis, 30 
Punctate basophilia of Grawitz, 553 
Purdy's method for albumin, 288 

for chlorids, 203 

for glucose, 315 

for phosphates, 210 

for sulphates, 215 
Purification of picric acid, 256 
Purin bases in urine, 196, 252, 359 
Purpura hemorrhagica, blood in, 593 
Purpurin, 268 
Purulent exudates, 740 

sputum, 5 

urine, 369 
Pus casts, 377 

cells in feces, 113 

in gastric contents, 62 
in sputum, 10 
in urine, 369 

enumeration of, 372 
significance of, 371 
tests for, 371 
Putrescin, 123, 360 
Putrid bronchitis, 32 

exudates, 740 
Pycnometer, 192, 438 
Pycnotic nucleus, 546 
Pyelitis, urine in, 369 

productiva, 266 
Pyelonephritis, 369 
Pyloric glands, 50 



Pyloric stenosis, 86, 92 
Pyogenic albumosuria, 294 
Pyonephrosis, 369 
Pyorrhea alveolaris, 42 
Pyrocatechin, 350 
Pyuria, 369 

Quantity of blood, 410 

of gastric juice, 57 

of urine, 180 
Quartan malarial parasite, 623, 627 
Quartan, asexual cycle of, 623 

sexual cycle of , 629 
Quotient, albumin, 289, 468 

protein, 289, 468 

volume, 414 

Ratio of N to CI output, 198 

to P2O5 output, 206 

to SO 3 output, 211 
Ray fungus, 27 
Reaction of blood, 418 

of feces, 121 

of gastric contents, 57 

of milk, 769 

of spinal fluid, 756 

of sputum, 3 

of urine, 187 
Reactivity of blood, 418 
Receptors, 656 

Rectum, blood in cancer of, 108 
Red cells (see Erythrocytes), 541 
in exudates, 739 
in feces, 109, 120 
in gastric contents, 59, 61, 83 
in sputum, 3, 5, 11 
in suspected stains, 727 
in urine, 372 

indigo, in urine, 272 

jigger, 168 

sputum, 3 
Refraction coefficient of serum, 467 
Reichmann's disease, 89 
Relapsing fever, 631 
Relative value of phosphoric acid, 206 
Removal of albumin, 288 

of glucose, 315, 317 

of turbidity, 281 
Renal abscess, 369 

albumosuria, 294 

aneurism, 373 

calculus, 385 

concretions, 385 

diabetes mellitus, 298, 394 

diagnosis, 388 

epistaxis, 373 

epithelial cells in urine, 367 

functional tests, 388 

hematuria, 373 

hemophilia, 373 

insufficiency, 388 
Rennin, 64, 81 
Resistance of red cells, 555 
Resorcin test for free HC], 67 
Rhabdonema intestinalis, 150 

strongyloides, 150 
Rhamnose in urine, 325 
Rheumatism, blood in, 614 
Rhinitis, 45 



INDEX 



837 



Rhizopoda in feces, 135 
Rice-water stools, 106, 129 

vomitus, 61 
Rickets, blood in, 606 
Rickett's organism of spotted fever, 646 
Riegel's method for chymosin, 81 

test meal, 55 
Rigg's disease, 42 
Ring bodies in red cells, 554 
of Cabot, 554 

worm of the beard, 169 
of the body, 169 

worm of the scalp, 170 
Ringers' -solution, 555 
Rivalta's test for exudates, 738 
Roberts' method for glucose, 321 
Roch's test for albumin, 285 
Rocky Mountain spotted fever, blood in, 

645 

organism of, 645 
Romanowsky's stain, 536 
Ronchese's method for ammonia, 244 
Rosacic acid, 268 

Rosenbach's method for bile pigments, 
348 

for skatoxyl, 272 
Rosenow's capsule stain, 652 
Rosin's test for bile pigments, 347 
Ross- J ones test of cerebrospinal fluid, 

763, 
Rot, grinders , 4 
Rouleaux formation, 541 
Round worms in feces, 148 
Rowntree and Geraghty's test, 394 
Rubner's test for lactose, 330 
Rudisch and Kleeberg's method for uric 

acid, 248 
Rudolf's method for coagulation time, 

441 
Ruhemann's uricometer, 251 
Russell and Brodie's coagulometer, 442 
Russo's test for typhoid, 353 
Rusty sputum, 3, 31 

Saccharometer of Einhorn, 320 

of Lohnstein, 320 
Saccharomyces cerevisiae, 15, 309 
Saccharose in urine, 331 
Sagitula hominis, 156 
Sago-like granules in sputum, i, il 
Sahli's desmoid reaction, 87 

hemometer, 460 

test-meal, 56 
Salicylic acid as preservative, 775 

test for gastric motility, 85 
Saliva, 35 

amount of, 35 

bacteria in, 36 

cells in, 36 

chemistry of, 35 

ferments in, 35 

microscopic examination of, 36 

nitrites in, 36 

obtaining of, 37 

pathologic changes in, 37 

potassium sulphocyanate in, 35 

ptyalin in, 35, 36 
Salivary corpuscles, 36 
Salivation, 37 



Salkowski-Ludwig method for uric acid, 

247 
Salol test of Ewald and Sievers, 85 
Salomon's test for gastric carcinoma, 93 
Salt glycosuria, 298 
Salzer's test-meal, 56 
Sand flea, 168 

intestinal, 118 

in urine, 385 

renal, 385 
Sanford's method for blood grouping, 

Sanguinous exudates, 739 

sputum, 5 
Saprophytes in feces, 128 

in sputum, 14 

in urine, 381 
Sarcinse in feces, 129 

in gastric contents, 63 

in sputum, 17 

in urine, 381 

ventriculi, 63 
Sarcoma, blood in, 617 
Sarcoptes scabiei, 165 
Saturation deficit, 73 
Scarlet fever, blood in, 610 
Schaer's test for blood, 729 
Scherer's method for albumin, 286 

test for leucin, 362 
Schick reaction, 667 
Schistocjrtes, 544 
Schistosomum hematobium in blood, 644 

_ in urine, 385 
Schizogone, 622 
Schizont, 622 

Schlosing's method for ammonia, 239 
Schmaltz' specific gravity tubes, 438 
Schmidt and Strasburger's standard diet, 

100 
Schmidt's fermentation method for feces, 

124 
Schiigner's granules, 626 
Schultze's granular cells, 560, 562 
Sclerostoma duodenale, 154 
Scybala, 105 
Seat worms, 149 
SebeHen's method for protein in milk, 

770 
Secondary anemia, 591 

proteoses, 293 

tubercular pleurisy, 750 
Secretin, 102 
Secretion of gastric juice, 51 

of genital organs, 398 

of mammary glands, 766 

of urine, 179 
Sedimentation, 354 
Sediments in urine, 354 

bacteria, 381 

bilirubin, 363 

blood cells, 372 

calcium carbonate, 367 
oxalate, 359 
phosphate, 364 
sulphate, 363 

casts, 374 

cholesterin, 364 

cystin, 360 

epithelial cells, 367 



838 



INDEX 



Sediments in urine, fat, 364 

hematoidin, 363 

hippuric acid, 363 

indigo, 271 

leucin, 361 

magnesium ammonium phosphate, 
366 
phosphate, 366 

mucoid material, 367 

mucous threads, 367 

nubecular threads, 184, 367 

organized, 367 

parasites, 384 

phosphates, 364, 365 

preservation of, 180 

pus cells, 369 

spermatozoa, 381 

tissue fragments, 381 

tyrosin, 362 

unorganized, 356 

urates, 357 

uric acid, 356 

xanthin, 359 
Sedimentum lateritium, 357 
SeliwanofT's test for levulose, 323 
Semen, 398 

chemistry of, 398 

medico-legal aspects of, 401 

microscopic examination of, 398 

pathology of, 400 

recognition of stains of, 401 

spermatic crystals in, 398 

spermatozoa in, 399 
Seminal stains, 401 

. medico-legal aspects of, 401 
Septic pleurisy, 750 
Sero diagnosis, 669 

of pregnancy, 703 

of syphilis, 680 

of typhoid, 670 
Serous cysts of the ovary, 752 

exudates, 738 

plethora, 412 

pleurisy, 750 
^ sputum, 5 . 

Serum albumin in blood, 466 
in urine, 274 

diagnosis of syphilis, 680 
of typhoid, 670 

globulin, 289 

determination of, 291 
significance of, 289 
test for, 290 
variations of, 289 

pathology, 655 

reactions, 669 

refraction coefficient of, 467 

special properties of, 655 
Sex, variations of blood cells due to, 

548 
Sexual cycle of malarial parasite, 629 

secretions, 398 
Shadows of leucocytes, 565 

red cells, 544 
Shaffer's method for acetone bodies, 345 
Shiga's bacillus, 132, 795 
Showers of casts, 375 
Side-chain theory of Ehrlich, 656 
Siderosis, 34 



Signet rings in malarial blood, 621, 625 
Significance of acetonuria, 334 

of albuminuria, 275 

of albumosuria, 294 

of Bence Jones proteinuria, 291 

of cylindruria, 375, 379 

of free HCl in gastric contents, 70 

of globulinuria, 289 

of glycosuria, 298 

of hematuria, 373 

of lactic acid in stomach, 74 

of leucocytosis, 568 

of levulosuria, 322 

of mucous threads in urine, 367, 378 

of mucus in feces, 1 1 1 

of nitrogen-partition of urine, 219 

of /3-oxybutyric acid in urine, 343 

of pentosuria, 325 

of pepsin in gastric juice, 78 

of pyuria, 369 
Sjoqvist's method for urea, 233 
Skatoxyl-potassium sulphate in urine, 

272 
Skin, blood in diseases of, 576 

parasites of, 164 
Sleeping sickness, blood in, 632 

organism of, 632 
Small-pox, blood in, 612 
Smears, preparation of, 526 

of blood, 526 

of exudates, 743, 748 

of feces, 119 

of pus, 743 

of sputum, 9 

of syphilitic material, 745 
Sntegma bacillus in buccal secretions, 37 
in exudates, 743 
in sputum, 24 
in urine, 382 
_ preputii, 743 
Smith's test for bile pigments, 347 
Soaps in feces, 116 
Sodium acid urate, 357 

carbonate as preservative of milk, 

774 

chlorid retention, 198 
in nephritis, 199, 391 
in pneumonia, 198 

in blood, 503 

in urine, 216 
Soft chancre, organism of, 744 
Soluble starch, 36, 82 
Solvents for blood stains, 728 
Specific gravity of blood, 437 

of cerebrospinal fluid, 756 

of exudates, 738 

of milk, 768 

of serum, 437 

of transudates, 737 

of urine, 192 
Specificity of agglutination test, 675I 

of complement-fixation test, 698 

of precipitin test, 679 
Spectrophotometer of Hiifner, 455 
Spectroscopic examination, 732 

tests for blood, 449, 732 
Spermatic crystals, 398 
Spermatocele, 754 
Spermatorrhea, 381, 398 



INDEX 



839 



Spermatozoa, 381, 399 
Spermin crystals, 14, 398 
Spiegler's test for albumin, 285 
Spirals of Curschmann, 7, 30, 31 
Spirillum of Asiatic cholera, 61, 129, 

799 

of Obermeier, 63 1 

of relapsing fever, 63 1 

of Vincent, 40 
Spirocheta, buccalis, 36 

icterohemorrhagica, 643 

nodosa, 644 

pallida, 636, 744 

characteristics of, 636, 638 

cultivation of, 639 

in blood, 638 

in exudates, 744 

in tissues, 746 

staining of, 636, 745 

refringens, 638 
Spironema balanitidis, 744 

recurrentis, 631 
Spit cups, 2 
Spleen, diseases of, blood in, 589 

removal of, 618 
Splenectomy, blood after, 618 
Splenic anemia, 589 
Splenocytes, 559 
Splenomegaly, blood in, 589 

tropical, 634 
Splenomyelogenous leukemia, 596 
Spore cyst, 629 
Spores, staining of, 789 
Sporoblast, 629 
Sporogone, 622 

Sporogony of malarial parasite, 629 
Sporothrix Schenckii, 174 
Sporotrichosis, 174 
Sporozoa in feces, 139 
Sporozoits, 629 

Spotted fever, organism of, 645 
Sputum, I 

air in, 4 

albumin in, 5 

amount of, 2 

bacteria in, 14 

biliary pigments in, 3 

blood in, 3, 5, 11 

character of, 4 

cheesy particles in, 6 

chemistry of, 5 

chromogenic bacteria in, 4 

coal pigment in, 4, 34 

coctum, 32 

collection of, 2 

color of, 3 

concretions in, 8 

consistency of, 2 

cotton fibers in, 4 

crudum, 31 

crystals in, 13 

Curschmann's spirals in, 7, 30, 31 

cytology of, 10 

deportment on standing of, 5 

Dittrich's plugs in, 7 

echinococcus membranes in, 9 

elastic tissue in, 12 

epithelial cells in, 1 1 

extraneous matter in, 9 



Sputum, fatty acids in, 13 

ferments in, 6 

ferric oxid in, 4 

fibrinous casts in, 8 

flour in, 4 

foreign bodies in, 9 

fundum petens, 4 

heart disease, cells in, 11, 32 

hemoglobin derivatives in, 11, 13 

in abscess of the lung, 33 

in actinomycosis, 27 

in acute bronchitis, 31 

in bronchial asthma, 33 

in broncho-pneumonia, 3I 

in chronic bronchitis, 32 

in croupous pneumonia, 31 

in fibrinous bronchitis, 32 

in gangrene of the lung, 33 

in influenza, 33 

in jaundice, 4 

in perforating empyema, 34 

in pneumonoconioses, 34 

in pulmonary tuberculosis, 30 

in putrid bronchitis, 32 

leucocytes in, 10 

macroscopic examination of, 6 

microscopic examination of,'(9 

morning, i 

mucin in, 5 

mucoid, 5 

mucopurulent, 5 

myelin granules in, 1 1 

nitrogen of, 2 

nummular, 5, 6 

odor of, 4 

origin of, i 

parasites in, 28 

prune-juice, 3, 31 

purulent, 5 

pus cells in, 10 

reaction of, 3 

red blood cells in, 1 1 

sanguinous, 5 

serous, 5 

spit-cups for, 2 

stone dust in, 4 

tenacity of, 3 

types of, 5 
Staining characteristics of tubercle ba- 
cillus, 20 

methods, principles of, 531 

of bacteria, 787 

of blood smears, 533 

of casts, 375 

of elastic tissue, 12 

properties of cells, 552 

vital, 540 
Stains, blood, 533 

Bunge and Trantenroth's, 24 

Burri's, 745 

dahlia, 563 

Ehrlich's tri-acid, 535 
triple, 535 

eosin hematoxylin, 534 
methylene blue, 533 

Fontana's, 746 

Gabbet's, 21 

Ghoreyeb's, 745 

Giemsa's, 538, 745 



840 



INDEX 



Stains, Goldhorn's, 638 

Gram's, 742 

india-ink, 745 

iodine, 563 

Jenner's, 536 

Leiner's, 115 

Leishman's, 537 

Levaditi and Manouelian's, 746 

Loffler's methylene blue, 21 

Mallory's, 176 

May-Griinwald's, 537 

Miillern's, 533 

Neisser's, 39 

Nocht's, 539 

Noguchi's, 747 

orcein, 12 

osmic acid, 116 

Pappenheim's blood, 536, 539 

for tubercle bacillus, 21 

polychrome, 536 

Romanowsky's, 536 

safranin, 743 

scharlach R, 116 

seminal, 401 

Sudan III, 116 

thionin, 112, 539 

Tribondeau's, 746 

Tunnicliff's, 745 

Tiirk's iodine, 563 

Unna-Tanzer's, 12 

Van Gieson's, 176 

Weigert's fibrin, 8 

Williams and Lowden's, 176 

Wright's, 537 

Zenoni's, 5 

Ziehl-Neelsen, 20 
Staphylococcus pyogenes in sputum, 26 
Starch in feces, 124 

detection of, 124 

digestion of, 36 

estimation of, 124 
Steatorrhea, 115, 123 
Stegomyia calopus, 642 

fasciata, 641 
Stercobilin, 107, 268 
Sterility, 400 
Sterilization, 779 
Stippled cells, 553, 611 
Stock-vaccines, 809 
Stomach, absorptive power of, 86 

carcinoma of, 91 

contents, 50 

dilatation of, 84, 85 

diseases of, 88 

ectasia of, 85 

fasting, 60 

function of, 84 

histology of, 50 

inflammation of, 90 

motihty of, 84 

tube, 52 

ulcer of, 91 

washing, 93 
Stomatitis, catarrhal, 40 

gonorrheal, 41 

mycotic, 41 

ulcerative, 40 

ulceromembranous, 40 
Stone cutters' phthisis, 4 



Stones, bronchial, 8 

gall, 117 

in bladder, 385 

in kidney, 385 

in lung, 8 

intestinal, 118 

in ureter, 385 

in urine, 385 

nasal, 46 

renal, 385 

ureteral 385 

vesical, 385 
Stools (see Feces), 99 

acholic, 107, 116 

clay- colored, 107 

curds in, 115 

frequency of, 104 

pea-soup, 130 

rice-water, 106, 129 
Strasburger's method for bacteria in 

feces, 128 
Strauss' test for lactic acid, 75 
Streptococcic sore throat, 41 
Streptococcus hemolyticus, 653, 802 

mucosus capsulatus, 653, 802 

pleurisy, 750 

pyogenes in sputum, 26 
in blood, 653 

viridans in blood, 653 
Streptothricosis, 27 
Streptothrix eppingeri, 14 
Striatula, 156 

Strongyloides intestinalis, 150 
Strongylus duodenalis, 154 

gigas, 163 

quadridentatus, 154 

renalis, 163 
Structural albuminuria, 278 
Sudanophiles, 565 
Sugar broth, 782 
Sugar-free broth, 782 
Sulphates of urine, 210 

easily split, 210 

ethereal, 211, 212 

preformed, 211 

total, 212 

unoxidized, 211 
Sulph-hemoglobin, 451 
Sulphhemoglobinemia, 451 
Sulphocyanates in saliva, 3^ 
Sulpho-salicylic acid test for albumin, 

285 
Sulphur compounds in urine, 210 

amount of, 210 

determination of, 213 

neutral, 213 

test for bile in urine, 349 

types of, 210 

variations of, 211 
Surgical interference, blood after, 603 
Syphilis, albuminuria of, 279 

blood in, 615 

hemoglobin test of Justus in, 616 

organism of, 636, 744 

serum test of Wassermann in, 683 

Tabanus striatus fabricus, 634 
Table for examination of calculi, 387 
Gaffky's, 23 



INDEX 



841 



Taeniidae in feces, 143 
tagnia asgyptica, 144 

canina, 144 

cucumerina, 144 

cucurbitina, 143 

dentata, 143 

diminuta, 145 

echinococcus, 145 

elliptica, 144 

flavopunctata, 145 

inermis, 143 

lata, 146 

leptocephala, 145 

mediocanellata, 143 

minima, 145 

moniliformis, 144 

nana, 144 

saginata, 143 

solium, 143 

varerina, 145 
Tallqvist's hemoglobinometer, 463 
Tape-worms in feces, 141 
Tartar of the teeth, 38 
Taurocholic acid, 117, 196, 349 
Taylor's ash-free diet, 200 
Teichmann's crystals, 731 

test for blood, 731 
Tenacity of sputum, 3 
Tertian malarial orgnism, 620, 626 
asexual cycle of, 622 
sexual cycle of, 629 
Test meal of Boas, 55 

of Ewald, 54 

of Fischer, 55 

of Riegel, 55 

of SahH, 56 

of Salzer, 56 

oil, 102 

renal, 391 
Testicular casts, 400 
Thecosoma hematobium, 646 
Theobromin, 252 
Theophyllin, 252 
Therapeutic measures, effect of, on blood, 

550, 573 
Thermolabile substances, 658 
Thermostable substances, 658 
Thionin stain, 112, 539 
Thiosulphuric acid in urine, 196 
Third corpuscles of blood, 580 
Thomas and Weber's method for pepsin, 

80 
Thoma-Zeiss hemocytometer, 510 
Thorn-apple crystals, 365 
Threads, mucus in urine, 184, 367, 378 
Thread worm, 149 
Threshold of chlorid excretion, 504 
Throat cultures, 39 
Thrombase, 440 
Thrush, 41 
Tick fever, 645 
Tide, alkaline, of urine, 191 
Timothy bacillus in sputum, 24 
Tinea barbae, 169 
circinata, 169 
favosa, 168 
sycosis, 169 
tonsurans, 170 
versicolor, 172 



Tissue, elastic in sputum, 12 
fragments in feces, 118 
in gastric contents, 63 
in sputum, 9 
in urine, 381 
Toisson's fluid, 514 
Tollen's orcin test for pentose, 327 

naphthoresorcin test for glycuronic 

acid, 333 ■ 
phloroglucin test, 326 
test for pentose, 326 
Tongue, coating of, 38 
Tonsillitis, leucocytosis in, 571 
Topfer's method for combined HCl, 72 
for free HCl, 68 
test for free HCl, 66 
Total acidity of gastric juice, 64 
components of, 64 
determination of, 64 
limits of, 65 
of urine, 188 
nitrogen of blood, 469 
of feces, 122 
of gastric juice, 82 
in carcinoma, 93 
of urine, 218 
amount of, 218 
determination of, 222 
variations of, 220 
solids of blood, 448 
of feces, 121 
of milk, 769 
of urine, 193, 196 
non-protein nitrogen of blood, 469 
volume of blood, 41 o 
Towel test for hemoglobin, 455 
Toxemia, hepatic, 229, 301, 322 
intestinal, 123 
renal, 388 
Toxogenic protein decomposition, 221 
Toxoids, 657 
Toxones, 657 
Toxophore, 657 
Trachoma bodies, 48 
Trachyochromatic erythroblasts, 545 
Transfusion, tests before, 723 
Transitional leucocytes, 559 
Transudates, 737 

coagulation of, 739 
obtaining of, 737 
properties of, 737 
Traumatic albuminuria, 279 
Treatment, effect of on Wassermann's 

test, 700 
Trematodes, 159 
in feces, 147 
in sputum, 29 
Treponema pallidum (see Spirocheta 

pallida), 636, 744 
Triacid stain of Ehrlich, 535 

of Pappenheim, 536 
Tribondeau's stain, 746 
Trichina spiralis, 152 
Trichinella spiralis, 152 
Trichinosis, 152, 576 
Trichiuris trichiura, 152 
Trichocephalus, dispar, 152 
hominis, 152 
mastigodes, 152 



842 



INDEX 



Trichocephalus, trichiuris, 152 
Trichomonas hominis, 140 

intestinalis, 140 

vaginalis, 384 
in urine, 384 
Trichophyton megalosporon endothrix, 
169 

microsporon, 170 
Trichotrachehdas, 152 
Triple phosphates as calculi, 388 

in sputum, 13 

in urine, 366 
Tripperfaden in urine, 380, 743 
Trombidiosis, 166 
Trombidium irritans, 165 
Trommer's test for glucose, 303 
Tropeolin test for HCl, 67 
Tropical splenomegaly, 634 
Tropics, anemia of, 548 
Trousseau's test for bile pigments, 347 
True albuminuria, 275 
Trypanosoma Brucei, 634 

equiperdum, 634 

Evansi, 634 

Gambiense, 632 
in the blood, 632 
in spinal fluid, 633, 760 
Trj^panosomiasis, 632 
Trypsin in feces, 102 

in pancreatic cysts, 754 

in luine, 265 
Tryptophan test, 93 
Tsetse flies, 632 

Tsuchiya's method for albumin, 287 
Tube casts in iirine, 374 
Tubercle bacilli in the blood, 654 

in exudates, 741 
inoscopy, 741 

in feces, 132 

in sputum, 17 

culture medium for, 19 

morphology of, 21 

number of, 23 

staining of, 20 

value of examination for, 22 

in urine, 382 
Tubercular meningitis, 760 

pleurisy, 750 
Tuberculin, 18 

reactions, 663 
Tuberculosis, blood in, 615 

complement-fixation test in, 702 

of bladder, 382 

of intestine, 132 

of kidneys, 370 

of lymph glands, 602 

of meninges, 760 

of peritoneum, 751 

of pleura, 750 

pulmonary, blood in, 615 
sputum in, 30 
Tuberculous cystitis, 382 
Tubular insufficiency, 388 
Tumor shreds in feces, 118 

in gastric contents, 63 

in urine, 381 
Tunnel workers' anemia, 156 ' 
Tunnicliff's bacillus, 45 

stain for spirochaete, 745 ^ 



Turk's iodin stain, 563 

counting chamber, 513 
Two-glass test, 371 
Types of pneumococci, 26 
Typhoid bacillus in the blood, 648 
in feces, 130 

Drigalski and Conradi's media, 

131 
Kendall and Day's media, 132 
in urine, 384 
fever, blood in, 609, 670 
feces in, 130 
Widal reaction in, 670 
pleurisy, 751 
Typhoidin reaction, 667 
Tyrosin in sputum, 13 
in urine, 259, 362 

Uffelmann's test for lactic acid, 75 
Uhlenhuth's antiformin method for tu- 
bercle bacilli, 18 
Ulcer of the stomach, 91 
Ulceromembranous angina of Vincent, 

40. 

Unaltered bile in feces, 108 
Uncinaria Americana, 155 

duodenalis, 155 
Uncinariasis, 154 

Undetermined nitrogen of urine, 259 
Unilateral nephritis, 396 
Unit of counting chamber, 518 
Unna-Tanzer's stain, 12 
Unorganized sediments in urine, 356 
Unoxidized sulphur of the urine, 213 
Uranium method for phosphates, 207 
Urates in urine, 357 
Urea in blood, 474 

in urine, 228 
amount of, 229 
determination of, 229 
variations of, 229 
Urease method for urea, 234, 474 
Uremia, blood in, 477 

urine in, 388 
Uremic coefficient, 477 

constant, 477 
Ureometer of Doremus, 230 

of Hinds, 221 
Ureteral calculi, 385 
Ureteritis membranacea, 266 
Urethritis, anterior, 371 

posterior, 371 
Uric acid, 242, 481 

calculi, 386 

diathesis, 245 

in blood, 481 

in the urine, 242 

determination of, 246 
metabolism of, 242 
variations of, 244 

sediment, 356 
Uricacidemia, 481 
Urine, 178 

acetone in, 196, 337 

acidity of, 188 

albumin in, 274 

albumoses in, 290, 293 

alkaline tide of, 191 

alkapton bodies in, 187, 350 



INDEX 



843 



Urine, alloxur bodies in, 196, 252, 359 
amino-acids in, 259 
ammonia in, 237 
amount of, 180 
animal gum in, 331 
animal parasites in, 384 
appearance of, 183 
ash of, 196 
bacteria in, 384 
Bence-Jones protein in, 290 
bile acids in, 349 
biliary pigments in, 346 
black, 186 
blood cells in, 372 
pigment in, 346 
blue, 186, 271 
calcium in, 216 
calculi in, 385 
carbohydrates in, 298 
carbonates in, 216, 367 
casts in, 374 

changes on standing of, 184 
chemistry of, 195 
chlorids in, 197 
cholesterin in, 364 
chromogens in, 267 
chyle in, 184, 364 
clearing of, 315, 317 
collection of, 179 
color of, 184 
composition of, 195 
consistence of, 184 
creatin in, 253, 258 
creatinin in, 253 
cryoscopy of, 390 
cultures of, 381 
cystin in, 213, 360,, 388 
dextrin in, 331 
dextrose in, 298 
diacetic acid in, 341 
diastase in, 265 
drug reactions in, 186 
Ehrlich's benzaldehyde reaction in, 

354 
diazo reaction in, 352 
egg-yellow reaction in, 353 

electric conductivity of, 390 

epithelial cells in, 367 

fat in, 364 

fatty acids in, 262 

ferments in, 264 

fibrin in, 297 

foreign bodies in, 384 

free mineral acidity of, 190 
organic acidity of, 190 

functional diagnosis from, 388 

glucose in, 298 

glycosuric acid in, 351 

glycuronic acid in, 331 

green, 186 

hematoporphyrin in, 346 

hemoglobin in, 295, 346 

hippuric acid in, 261, 363 

homogentisic acid in, 187, 351 

indican in, 270 

indigo in, 270 

inosite in, 331 

iron in, 218 

lactic acid in, 262 



Urine, lactose in, 329 

laiose in, 325 

leucin in, 259, 361 

leucocytes in, 369 

levulose in, 322 

magnesium in, 216 

maltose in, 330 

melanin in, 185, 350 

microscopy of, 354 

mucin-like substances in, 265 

mucoid material in, 367 

neutral sulphur in, 213 

nitrogen in, 218 

nitrogenous bodies in, 218 

nubecula in, 184, 367 

nuclein bodies in, 252 

nucleo-albumin in, 266 

odor of, 187 

optical activity of, 195 

organized sediments of, 367 ] 

oxalic acid in, 263 

oxaluric acid in, 263 

/3-oxybutyric acid in, 343 

parasites in, 384 

pentoses in, 325 

peptone in, 295 

phosphates in, 203, 364, 366 

physical properties of, 180 

pigments in, 267, 346 

potassium in, 216 

preservation of, 179 

protein of, 274 

proteoses in, 290 

ptomaines in, 360 

purin bases in, 252 

pus in, 369 

quantity of, 180 

reaction of, 187 

Russo's reaction in, 353 

sediments of, 356 

serum-albumin in, 274 
globulin in, 289 

skatoxyl in, 272 

sodium in, 216 

solids of, 193, 196 

specific gravity of, 192 

spermatozoa in, 381 

sugar in, 298 

sulphates in, 210 

sulphur compounds in, 210 

tissue fragments in, 381 

total solids of, 193, 196 

tyrosin in, 259, 362 

urates in, 357 

urea in, 228 

uric acid in, 242, 356 

urobilin in, 268 

urochrome in, 267 

uroerythrin in, 268 

urohematin in, 269 

urorosein in, 274 

xanthin bases in, 252, 359 
Urinod, 187 
Urinometer, 192 
Urinous odor 187 
UrobiHn, 268 
Urobilinuria, 268 
Urochrome, 267 
Urochromogen, Weisz' test for, 267 



844 



INDEX 



Uroerythrin, 268 
Uroferric acid, 213 
Uroleucic acid, 196, 351 
Urophain, 274 
Urorhodin, 272 
Uroroseinogen, 274 
Urorubin, 272 
Urostealith calculi, 388 
Uterine secretions, 405 

Vaccination, antityphoid, 676, 811 
Vaccines, 661, 808 

autogenous, 811 

diagnostic use of, 662 

preparation of, 809 

stock, 809 
Vaccine therapy, 661 
Vacuolization, 553 
Vaginal secretions, 402 
V^aginitis, catarrhal, 403 

gonorrheal, 404 
Value of blood examinations, 732 

of functional renal diagnosis, 388 

of search for tubercle bacilli, 22 
Van Deen's test for blood, 109, 728 
Van Ermengem's stain for flagella, 788 
Van Gieson's stain for Negri bodies, 176 
Van Slyke and Cullen's method for CO, 
capacity of blood, 422 

for urea, 234, 474 
Van Slyke and Donleavy's method for 

plasma chlorids, 505 
Van Slyke, Stillman and Cullen's titra- 
tion method, 426 
Vaquez' disease, 552 

Variations in number of leucocytes, 567, 
578 

of red cells, 548, 556 
Variola, blood in, 612 
Venous blood, 415 

puncture, 415, 647, 686 
Vermiculus, 629 
Vernal conjunctivitis, 49 
Vesicular albuminuria, 279 

mole, 406 
Vincent's angina, 40 

bacillus, 40 

spirillum, 40 
Vinegar eel in urine, 384 
Viscosity of blood, 439 
Vitali's test for pus, 371 
Vital staining of blood cells, 540 
Voges-Proskauer reaction, 794 
Volatile alkalinity of urine, 191 
Volhard's method for chlorids, 200 
Volume index of blood, 414 

of blood, 410 

quotient, 414 

value, 414 
Vomitus, 60 

bile in, 61 

blood in, 61 

fecal. 61 



Vomitus, green, 61 
mucus in, 61 
odor of, 61 

pancreatic fluid in, 61 
parasites in, 61 
pus in, 61 
rice water, 61 

Wagner's test for blood, 1 1 1 
Wang's method for indican, 272 
Warfield's test for pregnancy, 260 
Wassermann's serum reaction for syph- 

ihs, 680 
Waxy casts, 376 
Weber's test for blood, no 
Weidel's test for xanthin, 359 
Weil's disease, 643 
Weinstein's test for gastric carcinoma, 

93 
Weisz' test for urochromogen, 267 
Welch's gas bacillus, 133, 797 
Weyl's test for creatinin, 255 
Whetstone crystals of uric acid, 357 

of xanthin, 359 
Whip worm, 152 

White blood cells (see Leucocytes), 557 
Whooping cough, bacillus of, 791 

blood in, 613 
Widal reaction, 670 
Williams and Lowden's stain for Negri 

bodies, 176 
Williamson's blood test in diabetes, 605 
Winternitz' method for gastric motility, 

86 
Winternitz', Henry and McPhedrans' 

test for catalase, 579 
Wolff and Junghans' test, 95 
Wright's opsonic method, 660 

stain for blood smears, 537 

vaccine therapy, 661 
Wright and Kinnicutt's method for 
blood plates, 524 

Xanthin bases in feces, 123 

in urine, 196, 252, 341, 359 
calculi, 388 

Xerosis bacillus, 40 

Xylose in urine, 325 

Yaoita's method for ova, 134 
Yeast cells in feces, 126 

in gastric contents, 62, 63 

in sputum, 15 

in urine, 310 
Yellow fever, blood in, 641 

mosquito theory of, 641 

Ziehl-Neelsen method for tubercle ba- 
cilli, 20 
Zygotes, 629 

Zymogens in gastric juice, 77, 81 
Zymophore, 658 



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